Genetically modified cells and methods for converting (r)-methylmalonyl coa to (s)-methylmalonyl coa

ABSTRACT

Recombinant  Escherichia coli  host cells that comprise recombinant DNA expression vectors that drive expression of methylmalonyl CoA mutase from  Propionibacterium shermanii  or  Streptomyces cinnamonensis  as well as  Propionibacterium shermanii  epimerase can produce S-methylmalonyl CoA, a required substrate for the production of polyketides by most modular polyketide synthases not present in wild-type  E. coli  host cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent Ser. No. 10/829,897,filed Apr. 21, 2004, issuing on Nov. 6, 2007 as U.S. Pat. No. 7,291,486,which is a continuation of U.S. patent application Ser. No. 09/942,407,filed Aug. 29, 2001, now abandoned, which is a Divisional of U.S. patentSer. No. 09/699,136, filed Oct. 27, 2000, which claims priority under 35U.S.C. § 1.119(e) to U.S. Provisional Patent Application Ser. No.60/161,703, filed Oct. 27, 1999, and these applications are expresslyincorporated herein by reference in their entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was supported in part by SBIR Grant No. GM56575. TheUnited States government has certain rights in this invention.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application is being filed electronically via the USPTO EFS-WEBserver, as authorized and set forth in MPEP §1730 II.B.2(a)(A), and thiselectronic filing includes an electronically submitted sequence (SEQ ID)listing. The entire content of this sequence listing is hereinincorporated by reference for all purposes. The sequence listing isidentified on the electronically filed .txt file as follows:

File Name Date of Creation Size (bytes) 300622004902Seqlist.txt Nov. 5,2007 3,844 bytes

FIELD OF THE INVENTION

The present invention provides recombinant methods and materials forproducing polyketides by recombinant DNA technology. The inventionrelates to the fields of agriculture, animal husbandry, chemistry,medicinal chemistry, medicine, molecular biology, pharmacology, andveterinary technology.

BACKGROUND OF THE INVENTION

Polyketides represent a large family of diverse compounds synthesizedfrom 2-carbon units through a series of condensations and subsequentmodifications. Polyketides occur in many types of organisms, includingfungi and mycelial bacteria, in particular, the actinomycetes. There area wide variety of polyketide structures, and the class of polyketidesencompasses numerous compounds with diverse activities. Erythromycin,FK-506, FK-520, megalomicin, narbomycin, oleandomycin, picromycin,rapamycin, spinocyn, and tylosin are examples of such compounds. Giventhe difficulty in producing polyketide compounds by traditional chemicalmethodology, and the typically low production of polyketides inwild-type cells, there has been considerable interest in findingimproved or alternate means to produce polyketide compounds. See PCTpublication Nos. WO 93/13663; WO 95/08548; WO 96/40968; 97/02358; and98/27203; U.S. Pat. Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639;5,672,491; 5,712,146; and 5,962,290; and Fu et al., 1994, Biochemistry33: 9321-9326; McDaniel et al., 1993, Science 262: 1546-1550; and Rohr,1995, Angew. Chem. Int. Ed. Engl. 34(8): 881-888, each of which isincorporated herein by reference.

Polyketides are synthesized in nature by polyketide synthase (PKS)enzymes. These enzymes, which are complexes of multiple large proteins,are similar to the synthases that catalyze condensation of 2-carbonunits in the biosynthesis of fatty acids. PKS enzymes are encoded by PKSgenes that usually consist of three or more open reading frames (ORFs).Two major types of PKS enzymes are known; these differ in theircomposition and mode of synthesis. These two major types of PKS enzymesare commonly referred to as Type I or “modular” and Type II “iterative”PKS enzymes. A third type of PKS found primarily in fungal cells hasfeatures of both the Type I and Type II enzymes and is referred to as a“fungal” PKS enzymes.

Modular PKSs are responsible for producing a large number of 12-, 14-,and 16-membered macrolide antibiotics including erythromycin,megalomicin, methymycin, narbomycin, oleandomycin, picromycin, andtylosin. Each ORF of a modular PKS can comprise one, two, or more“modules” of ketosynthase activity, each module of which consists of atleast two (if a loading module) and more typically three (for thesimplest extender module) or more enzymatic activities or “domains.”These large multifunctional enzymes (>300,000 kDa) catalyze thebiosynthesis of polyketide macrolactones through multistep pathwaysinvolving decarboxylative condensations between acyl thioesters followedby cycles of varying β-carbon processing activities (see O'Hagan, D. Thepolyketide metabolites; E. Horwood: New York, 1991, incorporated hereinby reference).

During the past half decade, the study of modular PKS function andspecificity has been greatly facilitated by the plasmid-basedStreptomyces coelicolor expression system developed with the6-deoxyerythronolide B (6-dEB) synthase (DEBS) genes (see Kao et al.,1994, Science, 265: 509-512, McDaniel et al., 1993, Science 262:1546-1557, and U.S. Pat. Nos. 5,672,491 and 5,712,146, each of which isincorporated herein by reference). The advantages to this plasmid-basedgenetic system for DEBS are that it overcomes the tedious and limitedtechniques for manipulating the natural DEBS host organism,Saccharopolyspora erythraea, allows more facile construction ofrecombinant PKSs, and reduces the complexity of PKS analysis byproviding a “clean” host background. This system also expeditedconstruction of the first combinatorial modular polyketide library inStreptomyces (see PCT publication Nos. WO 98/49315 and 00/024907, eachof which is incorporated herein by reference).

The ability to control aspects of polyketide biosynthesis, such asmonomer selection and degree of β-carbon processing, by geneticmanipulation of PKSs has stimulated great interest in the combinatorialengineering of novel antibiotics (see Hutchinson, 1998, Curr. Opin.Microbiol. 1: 319-329; Carreras and Santi, 1998, Curr. Opin. Biotech. 9:403-411; and U.S. Pat. Nos. 5,712,146 and 5,672,491, each of which isincorporated herein by reference). This interest has resulted in thecloning, analysis, and manipulation by recombinant DNA technology ofgenes that encode PKS enzymes. The resulting technology allows one tomanipulate a known PKS gene cluster either to produce the polyketidesynthesized by that PKS at higher levels than occur in nature or inhosts that otherwise do not produce the polyketide. The technology alsoallows one to produce molecules that are structurally related to, butdistinct from, the polyketides produced from known PKS gene clusters.

There has been a great deal of interest in expressing polyketidesproduced by Type I and Type II PKS enzymes in host cells that do notnormally express such enzymes. For example, the production of the fungalpolyketide 6-methylsalicylic acid (6-MSA) in heterologous E. coli,yeast, and plant cells has been reported. See Kealey et al., January1998, Production of a polyketide natural product innonpolyketide-producing prokaryotic and eukaryotic host, Proc. Natl.Acad. Sci. USA 95:505-9, U.S. Pat. No. 6,033,883, and PCT PatentPublication Nos. 98/27203 and 99/02669, each of which is incorporatedherein by reference. Heterologous production of 6-MSA required or wasconsiderably increased by co-expression of a heterologous acyl carrierprotein synthase (ACPS) and that, for E. coli, media supplements werehelpful in increasing the level of the malonyl CoA substrate utilized in6-MSA biosynthesis. See also, PCT Patent Publication No. 97/13845,incorporated herein by reference.

The biosynthesis of other polyketides requires substrates other than orin addition to malonyl CoA. Such substrates include, for example,propionyl CoA, 2-methylmalonyl CoA, 2-hydroxymalonyl CoA, and2-ethylmalonyl CoA. Of the myriad host cells possible for utilization aspolyketide producing hosts, many do not naturally produce suchsubstrates. Given the potential for making valuable and usefulpolyketides in large quantities in heterologous host cells, there is aneed for host cells capable of making the substrates required forpolyketide biosynthesis. The present invention helps meet that need byproviding recombinant host cells, expression vectors, and methods formaking polyketides in diverse host cells.

SUMMARY OF THE INVENTION

The present invention provides recombinant host cells and expressionvectors for making products in host cells that are otherwise unable tomake those products due to the lack of a biosynthetic pathway to producea precursor required for biosynthesis of the product. The presentinvention also provides methods for increasing the amounts of a productproduced in a host cell by providing recombinant biosynthetic pathwaysfor production of a precursor utilized in the biosynthesis of a product.

In one embodiment, the host cell does not produce the precursor, and thehost cell is modified by introduction of a recombinant expression vectorso that it can produce the precursor. In another embodiment, theprecursor is produced in the host cell in small amounts, and the hostcell is modified by introduction of a recombinant expression vector sothat it can produce the precursor in larger amounts. In a preferredembodiment, the precursor is a primary metabolite that is produced infirst cell but not in a second heterologous cell. In accordance with themethods of the invention, the genes that encode the enzymes that producethe primary metabolite in the first cell are transferred to the secondcell. The transfer is accomplished using an expression vector of theinvention. The expression vector drives expression of the genes andproduction of the metabolite in the second cell.

In a preferred embodiment, the product is a polyketide. The polyketideis a polyketide synthesized by either a modular, iterative, or fungalPKS. The precursor is selected from the group consisting of malonyl CoA,propionyl CoA, methylmalonyl CoA, ethylmalonyl CoA, and hydroxymalonylor methoxymalonyl CoA. In an especially preferred embodiment, thepolyketide utilizes methylmalonyl CoA in its biosynthesis. In onepreferred embodiment, the polyketide is synthesized by a modular PKSthat requires methylmalonyl CoA to synthesize the polyketide.

In one embodiment, the host cell is either a procaryotic or eukaryotichost cell. In one embodiment, the host cell is an E. coli host cell. Inanother embodiment, the host cell is a yeast host cell. In anotherembodiment, the host cell is an Actinomycetes host cell, including butnot limited to a Streptomyces host cell. In another embodiment, the hostcell is a plant host cell. In a preferred embodiment, the host cell iseither an E. coli or yeast host cell, the product is a polyketide, andthe precursor is methylmalonyl CoA.

In one embodiment, the invention provides a recombinant expressionvector that comprises a promoter positioned to drive expression of oneor more genes that encode the enzymes required for biosynthesis of aprecursor. In a preferred embodiment, the promoter is derived from a PKSgene. In a related embodiment, the invention provides recombinant hostcells comprising one or more expression vectors that drive expression ofthe enzymes that produce the precursor.

In another embodiment, the invention provides a recombinant host cellthat comprises not only an expression vector of the invention but alsoan expression vector that comprises a promoter positioned to driveexpression of a PKS. In a related embodiment, the invention providesrecombinant host cells comprising the vector that produces the PKS andits corresponding polyketide. In a preferred embodiment, the host cellis an E. coli or yeast host cell.

These and other embodiments of the invention are described in moredetail in the following description, the examples, and claims set forthbelow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the modules and domains of DEBS and the biosynthesis of6-dEB from propionyl CoA and methylmalonyl CoA.

FIG. 2 shows the construction of pSK-MUT, in which four PCR fragmentswere sequenced and pieced together to form the complete mutase gene inpSK-bluescript.

FIG. 3 shows the acyl-CoA analysis in BL21(DE3) panD strains in vivo.

FIG. 4 shows the results of CoA analysis of E. coli overexpressingmethylmalonyl-CoA mutase. The levels of ³H detected in fractionscollected from HPLC of cell-free extracts from ³H P-alanine-fed E. coliharboring either the pET control vector grown without hydroxocobalamin(solid trace), pET grown with hydroxocobalamin (dash-dotted trace), pEToverexpressing the mutase and grown without hydroxocobalamin (dottedtrace), or pET overexpressing the mutase and grown with hydroxocobalamin(dashed trace) are shown.

FIG. 5 shows the three routes or biosynthetic pathways for the synthesisof methylmalonyl-CoA that can be engineered into yeast.

FIG. 6 shows acyl-CoA analysis of E. coli overexpressingmethylmalonyl-CoA mutase. The levels of ³H detected in fractionscollected from HPLC of cell-free extracts from ³H P-alanine-fed E. coliharboring either the pET control vector (solid trace) or pEToverexpressing the mutase (dashed trace) is shown.

FIG. 7 shows acyl-CoA analysis in S. cerevisiae. The levels of ³Hdetected in fractions collected from HPLC of cell-free extracts from ³HP-alanine-fed S. cerevisiae after growth ofr 24 hours (solid trace), 48hours (dashed trace) and 66 hours (dotted trace) is shown.

FIG. 8 shows the Common Cloning Cassette.

FIG. 9 shows a general method for cloning genes into yeast expressionvectors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides recombinant host cells and expressionvectors for making products in host cells, which are otherwise unable tomake those products due to the lack of a biosynthetic pathway to producea precursor required for biosynthesis of the product. As used herein,the term recombinant refers to a cell, compound, or composition producedat least in part by human intervention, particularly by modification ofthe genetic material of a cell. The present invention also providesmethods for increasing the amounts of a product produced in a host cellby providing recombinant biosynthetic pathways for production of aprecursor utilized in the biosynthesis of a product.

In one embodiment, the host cell does not produce the precursor, and thehost cell is modified by introduction of a recombinant expression vectorso that it can produce the precursor. In another embodiment, theprecursor is produced in the host cell in small amounts, and the hostcell is modified by introduction of a recombinant expression vector sothat it can produce the precursor in larger amounts. In a preferredembodiment, the precursor is a primary metabolite that is produced infirst cell but not in a second heterologous cell. In accordance with themethods of the invention, the genes that encode the enzymes that producethe primary metabolite in the first cell are transferred to the secondcell. The transfer is accomplished using an expression vector of theinvention. The expression vector drives expression of the genes andproduction of the metabolite in the second cell.

The invention, in its most general form, concerns the introduction, inwhole or in part, of a metabolic pathway from one cell into aheterologous host cell. The invention also encompasses the modificationof an existing metabolic pathway, in whole or in part, in a cell,through the introduction of heterologous genetic material into the cell.In all embodiments, the resulting cell is different with regard to itscellular physiology and biochemistry in a manner such that thebio-synthesis, bio-degradation, transport, biochemical modification, orlevels of intracellular metabolites allow production or improveexpression of desired products. The invention is exemplified byincreasing the level of polyketides produced in a heterologous host andby restricting the chemical composition of products to the desiredstructures.

Thus, in a preferred embodiment, the product produced by the cell is apolyketide. The polyketide is a polyketide synthesized by either amodular, iterative, or fungal PKS. The precursor is selected from thegroup consisting of malonyl CoA, propionyl CoA, methylmalonyl CoA,ethylmalonyl CoA, and hydroxymalonyl or methoxymalonyl CoA. In anespecially preferred embodiment, the polyketide utilizes methylmalonylCoA in its biosynthesis. In one preferred embodiment, the polyketide issynthesized by a modular PKS that requires methylmalonyl CoA tosynthesize the polyketide.

The polyketide class of natural products includes members having diversestructural and pharmacological properties (see Monaghan and Tkacz, 1990,Annu. Rev. Microbiol. 44: 271, incorporated herein by reference).Polyketides are assembled by polyketide synthases through successivecondensations of activated coenzyme-A thioester monomers derived fromsmall organic acids such as acetate, propionate, and butyrate. Activesites required for condensation include an acyltransferase (AT), acylcarrier protein (ACP), and beta-ketoacylsynthase (KS). Each condensationcycle results in a β-keto group that undergoes all, some, or none of aseries of processing activities. Active sites that perform thesereactions include a ketoreductase (KR), dehydratase (DH), andenoylreductase (ER). Thus, the absence of any beta-keto processingdomain results in the presence of a ketone, a KR alone gives rise to ahydroxyl, a KR and DH result in an alkene, while a KR, DH, and ERcombination leads to complete reduction to an alkane. After assembly ofthe polyketide chain, the molecule typically undergoes cyclization(s)and post-PKS modification (e.g. glycosylation, oxidation, acylation) toachieve the final active compound.

Macrolides such as erythromycin and megalomicin are synthesized bymodular PKSs (see Cane et al., 1998, Science 282: 63, incorporatedherein by reference). For illustrative purposes, the PKS that producesthe erythromycin polyketide (6-deoxyerythronolide B synthase or DEBS;see U.S. Pat. No. 5,824,513, incorporated herein by reference) is shownin FIG. 1. DEBS is the most characterized and extensively used modularPKS system. DEBS synthesizes the polyketide 6-deoxyerythronolide B(6-dEB) from propionyl CoA and methylmalonyl CoA. In modular PKS enzymessuch as DEBS, the enzymatic steps for each round of condensation andreduction are encoded within a single “module” of the polypeptide (i.e.,one distinct module for every condensation cycle). DEBS consists of aloading module and 6 extender modules and a chain terminatingthioesterase (TE) domain within three extremely large polypeptidesencoded by three open reading frames (ORFs, designated eryAI, eryAII,and eryAIII).

Each of the three polypeptide subunits of DEBS (DEBSI, DEBSII, andDEBSIII) contains 2 extender modules, DEBSI additionally contains theloading module. Collectively, these proteins catalyze the condensationand appropriate reduction of 1 propionyl CoA starter unit and 6methylmalonyl CoA extender units. Modules 1, 2, 5, and 6 contain KRdomains; module 4 contains a complete set, KR/DHIER, of reductive anddehydratase domains; and module 3 contains no functional reductivedomain. Following the condensation and appropriate dehydration andreduction reactions, the enzyme bound intermediate is lactonized by theTE at the end of extender module 6 to form 6-dEB.

More particularly, the loading module of DEBS consists of two domains,an acyl-transferase (AT) domain and an acyl carrier protein (ACP)domain. In other PKS enzymes, the loading module is not composed of anAT and an ACP but instead utilizes a partially inactivated KS, an AT,and an ACP. This partially inactivated KS is in most instances calledKS^(Q), where the superscript letter is the abbreviation for the aminoacid, glutamine, that is present instead of the active site cysteinerequired for full activity. The AT domain of the loading modulerecognizes a particular acyl CoA (propionyl for DEBS, which can alsoaccept acetyl) and transfers it as a thiol ester to the ACP of theloading module. Concurrently, the AT on each of the extender modulesrecognizes a particular extender-CoA (methylmalonyl for DEBS) andtransfers it to the ACP of that module to form a thioester. Once the PKSis primed with acyl- and malonyl-ACPs, the acyl group of the loadingmodule migrates to form a thiol ester (trans-esterification) at the KSof the first extender module; at this stage, extender module 1 possessesan acyl-KS and a methylmalonyl ACP. The acyl group derived from theloading module is then covalently attached to the alpha-carbon of themalonyl group to form a carbon-carbon bond, driven by concomitantdecarboxylation, and generating a new acyl-ACP that has a backbone twocarbons longer than the loading unit (elongation or extension). Thegrowing polyketide chain is transferred from the ACP to the KS of thenext module, and the process continues.

The polyketide chain, growing by two carbons each module, issequentially passed as a covalently bound thiol ester from module tomodule, in an assembly line-like process. The carbon chain produced bythis process alone would possess a ketone at every other carbon atom,producing a poyketone, from which the name polyketide arises. Commonly,however, the beta keto group of each two-carbon unit is modified justafter it has been added to the growing polyketide chain but before it istransferred to the next module by either a KR, a KR plus a DH, or a KR,a DH, and an ER. As noted above, modules may contain additionalenzymatic activities as well.

Once a polyketide chain traverses the final extender module of a PKS, itencounters the releasing domain or thioesterase found at the carboxylend of most PKSs. Here, the polyketide is cleaved from the enzyme andtypically cyclyzed. The resulting polyketide can be modified further bytailoring or modification enzymes; these enzymes add carbohydrate groupsor methyl groups, or make other modifications, i.e., oxidation orreduction, on the polyketide core molecule. For example, the final stepsin conversion of 6-dEB to erythromycin A include the actions of a numberof modification enzymes, such as: C-6 hydroxylation, attachment ofmycarose and desosamine sugars, C-12 hydroxylation (which produceserythromycin C), and conversion of mycarose to cladinose viaO-methylation.

With this overview of PKS and post-PKS modification enzymes and theirsubstrates, one can better appreciate the benefits provided by thepresent invention. DEBS is produced naturally in Saccharopolysporaerythraea and has been transferred to a variety of Streptomyces species,such as S. coelicolor CH999 and S. lividans K4-114 and K4-155, in whichit functions without further modification of the host cell to produce6-dEB. Thus, S. erythraea, S. coelicolor, and S. lividans make therequired precursors for 6-dEB synthesis. However, many othernon-Saccharopolyspora, non-Streptomyces host cells do not make all ofthe required precursors or make them only at levels sufficient tosupport only very small amounts of polyketide biosynthesis.

The present invention provides recombinant DNA expression vectors andmethods for making a polyketide and its required precursors in any hostcell. In one embodiment, the host cell is either a procaryotic oreukaryotic host cell. In a preferred embodiment, the host cell is an E.coli host cell. In another preferred embodiment, the host cell is ayeast host cell. In another embodiment, the host cell is a plant hostcell. In a preferred embodiment, the host cell is either an E. coli oryeast host cell, the product is a polyketide, and the precursor ismethylmalonyl CoA.

The recombinant expression vectors of the invention comprise a promoterpositioned to drive expression of one or more genes that encode theenzymes required for biosynthesis of a precursor. In a preferredembodiment, the promoter is derived from a PKS gene. In anotherpreferred embodiment, the promoter is one derived from a host cell geneor from a virus or phage that normally infects the host cell and isheterologous to the gene that encodes the biosynthetic enzyme.

In another embodiment, the invention provides a recombinant host cellthat comprises not only an expression vector of the invention but alsoan expression vector that comprises a promoter positioned to driveexpression of a PKS. In a related embodiment, the invention providesrecombinant host cells comprising the vector that produces the PKS andits corresponding polyketide. In a preferred embodiment, the host cellis an E. coli or yeast host cell.

Neither E. coli nor yeast makes sufficient methylmalonyl CoA to supportbiosynthesis of large amounts of polyketides that require methylmalonylCoA in their biosynthesis, and most species do not produce themethylmalonyl CoA substrate at all. In one embodiment, the presentinvention provides E. coli, yeast, and other host cells that producemethylmalonyl CoA in amounts sufficient to support polyketidebiosynthesis. In preferred embodiments, the cells produce sufficientamounts of methylmalonyl CoA to support biosynthesis of polyketidesrequiring methylmalonyl CoA for their biosynthesis at levels rangingfrom 1 μg/L, to 1 mg/L, to 10 mg/L, to 100 mg/L, to 1 g/L, to 10 g/L.

In one embodiment, the host cells of the invention have been modified toexpress a heterologous methylmalonyl CoA mutase. This enzyme, whichconverts succinyl CoA to methylmalonyl CoA (although the reversereaction is 20 times more favored) has been expressed in E. coli using agene cloned from propionibacteria but was inactive due to the lack ofvitamin B12. In accordance with the methods of the present invention,this enzyme can be made in an active form in E. coli and other hostcells by either expressing (constitutively or otherwise) a B12transporter gene, such as the endogenous E. coli gene and/or byutilizing a media that facilitates B 12 uptake (as used herein, B12 canrefer to the precursor hydroxocobalamin, which is converted to B12).While certain methylmalonyl CoA mutases make the R-isomer, including themethylmalonyl CoA mutases derived from the propionibacteria, theR-isomer can be converted to the S-isomer using an epimerase. Forexample, epimerase genes from propionibacteria or Streptomyces can beemployed for this purpose.

In another embodiment, the host cells of the invention have beenmodified to express a heterologous propionyl CoA carboxylase thatconverts propionyl CoA to methylmalonyl CoA. In this embodiment, one canfurther increase the amount of methylmalonyl CoA precursor by culturingthe cells in a media supplemented with propionate. In a preferredembodiment, the host cells are E. coli host cells.

Thus, in accordance with the methods of the invention, the heterologousproduction of certain polyketides in E. coli, yeast, and other hostorganisms require both the heterologous expression of a desired PKS andalso the enzymes that produce at least some of the substrate moleculesrequired by the PKS. These substrate molecules, called precursors, arenot normally found as intracelluar metabolites in the host organism orare present in low abundance. The present invention provides a method toproduce or modify the composition or quantities of intracellularmetabolites within a host organism where such metabolites are notnaturally present or are present in non-optimal amounts.

A specific embodiment of the present invention concerns the introductionand modification of biochemical pathways for methylmalonyl CoAbiosynthesis. Methylmalonyl CoA, as noted above, is a substrate utilizedfor the synthesis of polyketides by many polyketide synthases. Some ofthe known biochemical pathways for the intracellular production ofmethylmalonyl CoA employ enzymes and their corresponding genes found incertain organisms. These enzymes and genes have not been found, or areotherwise non-optimal, in other organisms. These other organisms includethose that could otherwise be very useful as heterologous hosts for theproduction of polyketides. The present invention provides methods toengineer a host organism so that it contains a new or modified abilityto produce methylmalonyl CoA and/or to increase or decrease the levelsof methylmalonyl CoA in the host.

As noted above, two biochemical pathways involving methylmalonyl CoA areparticularly relevant to this aspect of the present invention. Thesepathways are the methylmalonyl CoA mutase pathway, hereafter referred toas the MUT pathway, and the propionyl CoA carboxylase pathway, hereafterreferred to as the PCC pathway.

The MUT pathway includes the enzymes methylmalonyl CoA mutase (5.4.99.2,using the numbering system devised by the Nomenclature Committee of theInternational Union of biochemistry and Molecular Biology),methylmalonyl CoA epimerase (5.1.99.1), and malonyl CoA decarboxylase(4.1.1.9). The biochemical pathway includes the conversion of succinylCoA to (R)-methylmalonyl CoA through the action of methylmalonyl CoAmutase (5.4.99.2) followed by the conversion of (R)-methylmalonyl CoA to(S)-methylmalonyl CoA through the action of methylmalonyl CoA epimerase(5.1.99.1). (S)-methylmalonyl CoA is a substrate utilized by severalpolyketide synthases. The enzyme malonyl CoA decarboxylase (4.1.1.9)catalyzes the decarboxylation of malonyl CoA but is also reported tocatalyze the decarboxylation of (R)-methylmalonyl CoA to form propionylCoA. Propionyl CoA is a substrate utilized by some polyketide synthases.

The PCC pathway includes the enzymes propionyl CoA carboxylase (6.4.1.3)and propionyl CoA synthetase (6.2.1.17). The biochemical pathwayincludes the conversion of propionate to propionyl CoA through theaction of propionyl CoA synthetase (6.2.1.17) followed by the conversionof propionyl CoA to (S)-methylmalonyl CoA through the action ofpropionyl CoA carboxylase (6.4.1.3). (S)-methylmalonyl CoA is thesubstrate utilized by many polyketide synthases.

An illustrative embodiment of the present invention employs specificenzymes from these pathways. As those skilled in the art will recognizeupon contemplation of this description of the invention, the inventioncan also be practiced using additional and/or alternative enzymesinvolved in the MUT and PCC pathways. Moreover, the invention can bepracticed using additional and alternative pathways for methylmalonylCoA and other intracelluar metabolites.

The methods of the invention involve the introduction of geneticmaterial into a host strain of choice to modify or alter the cellularphysiology and biochemistry of the host. Through the introduction ofgenetic material, the host strain acquires new properties, e.g. theability to produce a new, or greater quantities of, an intracellularmetabolite. In an illustrative embodiment of the invention, theintroduction of genetic material into the host strain results in a newor modified ability to produce methylmalonyl CoA. The genetic materialintroduced into the host strain contains gene(s), or parts of genes,coding for one or more of the enzymes involved in thebio-synthesis/bio-degradation of methylmalonyl CoA and may also includeadditional elements for the expression and/or regulation of expressionof these genes, e.g. promoter sequences. Specific gene sequences codingfor enzymes involved in the bio-synthesis/bio-degradation ofmethylmalonyl CoA are listed below.

A suitable methylmalonyl CoA mutase (5.4.99.2) gene can be isolated fromStreptomyces cinnamonensis. See Birch et al., 1993, J. Bacteriol. 175:3511-3519, entitled “Cloning, sequencing, and expression of the geneencoding methylmalonyl-coenzyme A mutase from Streptomycescinnamonensis.” This enzyme is a two subunit enzyme; the A and B subunitcoding sequences are available under Genbank accession L10064. Anothersuitable methylmalonyl CoA mutase gene can be isolated fromPropionibacterium shermanii. See Marsh et al., 1989, Biochem. J. 260:345-352, entitled “Cloning and structural characterization of the genescoding for adenosylcobalamin-dependent methylmalonyl CoA mutase fromPropionibacterium shermanii.” Alternatively, a suitable methylmalonylCoA mutase gene can be isolated from Porphyromonas gingivalis. SeeJackson et al., 1995, Gene 167: 127-132, entitled “Cloning, expressionand sequence analysis of the genes encoding the heterodimericmethylmalonyl CoA mutase of Porphyromonas gingivalis W50.”Alternatively, suitable methylmalonyl CoA mutase genes can be isolatedfrom any of the sources noted in the following table of a partial BLASTsearch report or from additional BLAST analyses.

Results of BLAST Search of NCBI Database for Methylmalonyl CoA MutasemutA gb|L10064|STMMUTA Streptomyces cinnamonensis 931 0.0 (querysequence) gb|AD000015|MSGY175 Mycobacterium tuberculosis sequence 3007e−80 emb|Z79701|MTCY277 Mycobacterium tuberculosis H37Rv 300 7e−80gb|AD000001|MSGY456 Mycobacterium tuberculosis sequence 238 8e−76emb|X14965|PSMUTAB Propionibacterium shermanii mutA 268 5e−70gb|L30136|POYMCMAB Porphyromonas gingivalis 137 9e−31gb|AE000375|AE000375 Escherichia coli K-12 MG1655 134 1e−29gb|U28377|ECU28377 Escherichia coli K-12 genome; 134 1e−29emb|X66836|ECSERAICI E. coli serA, iciA, sbm genes 133 1e−29gb|AF080073|SMPCAS2 Sinorhizobium meliloti 130 2e−28ref|NM_000255.1|MUT| Homo sapiens 113 2e−23 dbj|AP000006|AP000006Pyrococcus horikoshii OT3 110 2e−22 emb|AJ248285.1|CNSPAX03 Pyrococcusabyssi 109 3e−22 emb|X51941|MMMMCOAM Mouse mRNA 109 3e−22gb|AE000952|AE000952 Archaeoglobus fulgidus section 155 104 9e−21emb|AJ237976.1|SCO237976 Streptomyces coelicolor icmA gene 103 2e−20dbj|AP000062.1|AP000062 Aeropyrum pernix genomic DNA 102 3e−20gb|U67612|SCU67612 Streptomyces cinnamonensis coenzyme B12 98 7e−19gb|AE001015|AE001015 Archaeoglobus fulgidus section 92 97 1e−18emb|X59424|BFOF4 Bacillus firmus OF4 genes for ATP binding 82 7e−14 mutBgb|L10064|STMMUTA Streptomyces cinnamonensis 1379 0.0 (query sequence)gb|AD000001|MSGY456 Mycobacterium tuberculosis 1018 0.0emb|Z79701|MTCY277 Mycobacterium tuberculosis H37Rv 1017 0.0gb|AD000015|MSGY175 Mycobacterium tuberculosis sequence 1017 0.0emb|X14965|PSMUTAB Propionibacterium shermanii 996 0.0gb|L30136|POYMCMAB Porphyromonas gingivalis methylmalonyl 882 0.0ref|NM_000255.1|MUT| Homo sapiens methylmalonyl Coenzyme A 855 0.0emb|X51941|MMMMCOAM Mouse mRNA 32 0.0 gb|U28377|ECU28377 Escherichiacoli K-12 genome 798 0.0 gb|AE000375|AE000375 Escherichia coli K-12MG1655 798 0.0 emb|X66836|ECSERAICI E. coli serA, iciA, sbm genes 7970.0 gb|AF080073|SMPCAS2 Sinorhizobium meliloti 782 0.0gb|AE001015|AE001015 Archaeoglobus fulgidus 516  e−145dbj|AP000062.1|AP000062 Aeropyrum pernix genomic DNA 408  e−139emb|AJ248285.1|CNSPAX03 Pyrococcus abyssi complete genome 486  e−135dbj|AP000006|AP000006 Pyrococcus horikoshii OT3 genomic DNA 480  e−133gb|AE000952|AE000952 Archaeoglobus fulgidus section 155 467  e−130emb|Z35604.1|CEZK1058 Caenorhabditis elegans cosmid ZK1058 316  e−109emb|AJ237976.1|SCO237976 Streptomyces coelicolor icmA 377  e−103gb|U67612|SCU67612 Streptomyces cinnamonensis coenzyme 372  e−101emb|AL035161|SC9C7 Streptomyces coelicolor cosmid 9C7 359 2e−97gb|U28335|MEU28335 Methylobacterium extorquens 351 4e−95gb|AF008569|AF008569 Streptomyces collinus coenzyme 337 8e−91gb|U65074|ECU65074 Escherichia coli chromosome 275 3e−72gb|M37500|HUMMUT03 Human methylmalonyl CoA mutase 202 3e−50gb|AF178673.1|AF178673 Streptomyces cinnamonensis 183 1e−44emb|Z49936.1|CEF13B10 Caenorhabditis elegans cosmid F13B10 138 2e−41gb|M37499|HUMMUT02 Human methylmalonyl CoA mutase 112 4e−23dbj|AP000001.1|AP000001 Pyrococcus horikoshii OT3 genomic 106 2e−21emb|AJ248283.1|CNSPAX01 Pyrococcus abyssi complete genome 106 2e−21gb|M37503|HUMMUT06 Human methylmalonyl CoA mutase 101 7e−20gb|M37508|HUMMUT11 Human methylmalonyl CoA mutase 86 3e−15gb|M37509|HUMMUT12 Human methylmalonyl CoA mutase 80 3e−13gb|M37501|HUMMUT04 Human methylmalonyl CoA mutase 77 2e−12

Methylmalonyl CoA mutase requires vitamin B12 (adenosylcobalamin) as anessential cofactor for activity. One of the difficulties in expressingactive methylmalonyl CoA mutase in a heterologous host is that the hostorganism may not provide sufficient, if any, amounts of this cofactor.Work on the expression of methionine synthase, a cobalamin-dependentenzyme, in E. coli, a host that does not synthesize cobalamin, has shownthat it is possible to express an active cobalamin-dependent enzyme byincreasing the rate of cobalamin transport. See Amaratunga et al., 1996,Biochemistry 35: 2453-2463, entitled “A synthetic module for the metHgene permits facile mutagenesis of the cobalamin-binding region ofEscherichia coli methionine synthase: initial characterization of sevenmutant proteins,” incorporated herein by reference.

The methods of the present invention include the step of increasing theavailability of cobalamin for the heterologous expression of activemethylmalonyl CoA mutase in certain hosts, e.g. E. coli. In particular,these methods incorporate growing cells in a media that containshydroxocobalamin and/or other nutrients, as described in Amaratunga etal., supra. Additional methods for increasing the availability ofcobalamin include constitutive and/or over-expression of vitamin B12transporter proteins and/or their regulators.

A suitable methylmalonyl CoA epimerase (5.1.99.1) gene for purposes ofthe present invention can be isolated from Streptomyces coelicolor asreported in GenBank locus SC5F2A as gene SC5F2A. 13 (referred to here asEP5) or from S. coelicolor as reported in GenBank locus SC6A5 as geneSC6A5.34 (referred to here as EP6). See Redenbach et al., 1996, Mol.Microbiol. 21 (1), 77-96, entitled “A set of ordered cosmids and adetailed genetic and physical map for the 8 Mb Streptomyces coelicolorA3(2) chromosome,” incorporated herein by reference. To date, nobiochemical characterization of the proteins encoded by the genes EP5and EP6 has been carried out; thus, the present invention provides amethod for using these genes to provide methylmalonyl CoA epimeraseactivity to a host. That these genes encode proteins with methylmalonylCoA epimerase activity is supported by their homology to the sequence ofa 2-arylpropionyl CoA epimerase from rat. See Reichel et al., 1997, Mol.Pharmacol. 51: 576-582, entitled “Molecular cloning and expression of a2-arylpropionyl-coenzyme A epimerase: a key enzyme in the inversionmetabolism of ibuprofen,” and Shieh & Chen, 1993, J. Biol. Chem. 268:3487-3493, entitled “Purification and characterization of novel‘2-arylpropionyl CoA epimerases’ from rat liver cytosol andmitochondria.” Both rat 2-arylpropionyl CoA epimerase and methylmalonylCoA epimerase catalyze the same stereoisomeric inversion, but withdifferent chemical groups attached.

Biochemical characterization of a methylmalonyl CoA epimerase enzymepurified from Propionibacterium shermanii has been completed. SeeLeadlay, 1981, Biochem. J. 197: 413-419, entitled “Purification andcharacterization of methylmalonyl CoA epimerase from Propionibacteriumshermanii,” Leadlay & Fuller, 1983, Biochem. J. 213: 635-642, entitled“Proton transfer in methylmalonyl CoA epimerase from Propionibacteriumshermanii: Studies with specifically tritiated (2R)-methylmalonyl CoA assubstrate; Fuller & Leadlay, 1983, Biochem. J. 213: 643-650, entitled“Proton transfer in methylmalonyl CoA epimerase from Propionibacteriumshermanii: The reaction of (2R)-methylmalonyl CoA in tritiated water.”The DNA sequence of the gene coding for this enzyme fromPropionibacterium shermanii is provided by the present invention as SEQID NO: 1 in isolated and recombinant form and is incorporated intoexpression vectors and host cells of the invention. Suitablemethylmalonyl CoA epimerase genes can be isolated from a BLAST searchusing the P. shermanii sequence provided in Example 1, below. Preferredepimerases in addition to the P. shermanii epimerase include geneidentified by homology with the P. shermanii sequence located on cosmid8F4 from the S. coelicolor genome sequencing project and the B. subtilisepimerase described by Haller et al., 2000, Biochemistry 39 (16):4622-4629, incorporated herein by reference.

One can also make S-methylmalonyl CoA from R-methylmalonyl CoA utilizingan activity of malonyl CoA decarboxylase A, which convertsR-methylmalonyl CoA to propionyl CoA. As described above, propionyl CoAcan then be converted to S-methylmalonyl CoA by propionyl CoAcarboxylase. A suitable malonyl CoA decarboxylase (4.1.1.9) gene forpurposes of the present invention can be isolated from Saccharopolysporaerythraea as reported in Hsieh & Kolattukudy, 1994, J. Bacteriol. 176:714-724, entitled “Inhibition of erythromycin synthesis by disruption ofmalonyl-coenzyme A decarboxylase gene eryM in Saccharopolysporaerythraea.” Alternatively, suitable malonyl CoA decarboxylase genes canbe isolated from any of the sources noted in the following table ofBLAST search reports or by additional BLAST searches.

Results of BLAST Search of NCBI Database for Malonyl CoA DecarboxylaseMalonyl CoA decarboxylase (DC) gb|L05192|SERMALCOAD S. erythraea malonyl664 0.0 (query sequence) emb|AL022268|SC4H2 Streptomyces coelicolorcosmid 4H2 128 3e−28 emb|Z75555|MTCY02B10 Mycobacterium tuberculosisH37Rv 109 1e−22 gb|AD000018|MSGY151 Mycobacterium tuberculosis sequence109 1e−22 gb|AF141323.1|AF141323 Shigella flexneri SHI-2 95 5e−18emb|X76100|ECIUC E. coli plasmid iucA, iucB and iucC genes 92 3e−17emb|AL116808.1|CNS01DGW Botrytis cinerea strain T4 cDNA 88 5e−16gb|AF110737.1|AF110737 Sinorhizobium meliloti strain 2011 84 9e−15emb|AL109846.1|SPBC17G9 S. pombe chromosome II cosmid c17G9 71 7e−11gb|L06163|PSEAAC Pseudomonas fluorescens aminoglycoside 70 1e−10

A suitable propionyl CoA carboxylase (6.4.1.3) gene for purposes of thepresent invention can be isolated from Streptomyces coelicolor asreported in GenBank locus AF113605 (pccB), AF113604 (accA2) and AF113603(accA1) by H. C. Gramajo and colleagues. The propionyl CoA carboxylasegene product requires biotin for activity. If the host cell does notmake biotin, then the genes for biotin tranport can be transferred tothe host cell. Even if the host cell makes or transports biotin, theendogenous biotin transferase enzyme may not have sufficient activity(whether due to specificity constraints or other reasons) to biotinylatethe propionyl CoA carboxylase at the rate required for high levelprecursor synthesis. In this event, one can simply provide the host cellwith a sufficiently active biotin transferase enzyme gene, or if thereis an endogenous transferase gene, such as the birA gene in E. coli, onecan simply overexpress that gene by recombinant methods. Many additionalgenes coding for propionyl CoA carboxylases, or acetyl CoA carboxylaseswith relaxed substrate specificity that includes propionate, have beenreported and can be used as sources for this gene, as shown in thefollowing table.

Results of BLAST Search of NCBI Database for Propionyl CoA CarboxylasePropionyl CoA Carboxylase (pccB) gb|AF113605.1|AF113605 S. coelicolorpropionyl 1035 0.0 (query sequence) emb|X92557|SEPCCBBCP S. erythraeapccB, bcpA2, and orfX 800 0.0 emb|Z92771|MTCY71 Mycobacteriumtuberculosis H37Rv 691 0.0 dbj|AB018531|AB018531 Corynebacteriumglutamicum dtsR1 686 0.0 gb|U00012|U00012 Mycobacterium leprae cosmidB1308 686 0.0 dbj|AB018530|AB018530 Corynebacterium glutamicum dtsR gene612  e−174 gb|AE001742.1|AE001742 Thermotoga maritima section 54 610 e−173 emb|AJ002015|PMAJ2015 Propionigenium modestum mmdD 589  e−167dbj|AB007000|AB007000 Myxococcus xanthus MxppcB gene 588  e−166gb|L48340|MTBKATA Methylobacterium extorquens catalase 588  e−166gb|AE000952|AE000952 Archaeoglobus fulgidus section 155 572  e−162dbj|AP000005|AP000005 Pyrococcus horikoshii OT3 genomic 570  e−161emb|AJ248285.1|CNSPAX03 Pyrococcus abyssi complete genome 570  e−161emb|AL031124|SC1C2 Streptomyces coelicolor cosmid 1C2 563  e−159gb|L22208|VEIMCDC Veillonella parvula methylmalonyl CoA 558  e−157gb|AF080235|AF080235 Streptomyces cyanogenus landomycin 552  e−155emb|AJ235272|RPXX03 Rickettsia prowazekii strain Madrid E 545  e−153dbj|AB000886|AB000886 Sus scrofa mRNA for Propionyl CoA 539  e−152ref|NM_000532.1|PCCB| Homo sapiens propionyl Coenzyme A 538  e−151emb|X73424|HSPCCBA Homo sapiens gene for propionyl CoA 538  e−151gb|M14634|RATPCCB Rat mitochondrial propionyl CoA 535  e−150gb|S67325|S67325 propionyl CoA carboxylase beta subunit 531  e−149gb|U56964|CELF52E4 Caenorhabditis elegans cosmid F52E4 367  e−143emb|Z99116|BSUB0013 Bacillus subtilis complete genome 494  e−138dbj|D84432|BACJH642 Bacillus subtilis DNA, 283 Kb region 494  e−138gb|AF042099|AF042099 Sulfolobus metallicus putative 486  e−136emb|AL022076.1|MTV026 Mycobacterium tuberculosis H37Rv 483  e−135gb|L04196|PRSTRANSC Propionibacterium shermanii 383  e−104emb|AL023635.1|MLCB1243 Mycobacterium leprae cosmid B1243 356 1e−96emb|Z70692.1|MTCY427 Mycobacterium tuberculosis H37Rv 353 1e−95gb|L78825|MSGB1723CS Mycobacterium leprae cosmid B1723 DNA 319 4e−93gb|M95713|RERCOABETA Rhodococcus erythropolis 340 5e−92emb|Z99113|BSUB0010 Bacillus subtilis complete genome 325 2e−87gb|U94697|CCU94697 Caulobacter crescentus DNA topoisomerase 270 6e−71emb|Z95556|MTCY07A7 Mycobacterium tuberculosis H37Rv 253 9e−66emb|Y07660|MTACCBC M. tuberculosis accBC gene 231 6e−59emb|Z79700|MTCY10D7 Mycobacterium tuberculosis H37Rv 229 2e−58dbj|AB018557.1|AB018557 Streptomyces griseus cyaA gene 228 5e−58gb|U46844|MSU46844 Mycobacterium smegmatis catalase 209 2e−52emb|Z19555.1|CEF02A9 Caenorhabditis elegans cosmid F02A9 105 9e−51gb|M13573|HUMPCCB Human propionyl CoA carboxylase beta 194 5e−48gb|AF030576|AF030576 Acidaminococcus fermentans 170 9e−41emb|Y13917|BSY13917 Bacillus subtilis ppsE, yngL, yngK 149 2e−34emb|X69435|AFGCDA A. fermentans GCDA gene for 107 1e−21emb|Z82368|RPZ82368 R. prowazekii genomic DNA fragment 93 2e−17gb|AF025469|CELW09B6 Caenorhabditis elegans cosmid W09B6 78 5e−13gb|U87980|MRU87980 Malonomonas rubra putative IS-element 78 7e−13gb|AE001518|AE001518 Helicobacter pylori, strain J99 75 6e−12gb|AE000604.1|AE000604 Helicobacter pylori 26695 section 82 75 8e−12gb|U89347|ACU89347 Acinetobacter calcoaceticus malonate 74 1e−11emb|AL021961|ATF28A23 Arabidopsis thaliana DNA 61 2e−11gb|AE001591|AE001591 Chlamydia pneumoniae section 7 73 2e−11emb|Z46886|UMACCGEN U. maydis ACC gene for acetyl coa 71 1e−10gb|U86128|SSPCCB1 Sus scrofa propionyl CoA carboxylase B 70 2e−10emb|AJ006497|HSA006497 Homo sapiens PCCB gene, exons 11 70 2e−10gb|AE001301|AE001301 Chlamydia trachomatis section 28 69 5e−10gb|U32724|U32724 Haemophilus influenzae Rd section 39 68 8e−10gb|U04358|PSU04358 Pseudomonas syringae pv. syringae Y30 68 8e−10Propionyl CoA carboxylase (accA2) gb|AF113604.1|AF113604 S. coelicolorputative 1101 0.0 (query sequence) gb|AF113603.1|AF113603 Streptomycescoelicolor putative 1090 0.0 gb|AF126429.1|AF126429 Streptomycesvenezuelae JadJ 967 0.0 emb|Z92771|MTCY71 Mycobacterium tuberculosisH37Rv 758 0.0 emb|X92557|SEPCCBBCP S. erythraea pccB, bcpA2, and orfXgenes 753 0.0 emb|X92556|SEHGTABCP S. erythraea hgtA, bcpA1, and orf122753 0.0 gb|U00012|U00012 Mycobacterium leprae cosmid B1308 746 0.0emb|X63470|MLBCCPG M. leprae gene for biotin carboxyl 743 0.0gb|U35023|CGU35023 Corynebacterium glutamicum thiosulfate 695 0.0gb|U24659|SVU24659 Streptomyces venezuelae glucose 599  e−170gb|AE000742|AE000742 Aquifex aeolicus section 74 413  e−113gb|U67563|U67563 Methanococcus jannaschii section 105 405  e−111gb|L36530|MQSPYRCARB Aedes aegypti pyruvate carboxylase 400  e−110gb|AF132152.1|AF132152 Drosophila melanogaster clone 396  e−108gb|L09192|MUSMPYR Mus musculus pyruvate carboxylase 393  e−107gb|U36585|RNU36585 Rattus norvegicus pyruvate carboxylase 391  e−107gb|U32314|RNU32314 Rattus norvegicus pyruvate carboxylase 391  e−107gb|L14862|ANAACCC Anabaena sp. (PCC 7120) 49.1 kDa biotin 388  e−106gb|U59234|SPU59234 Synechococcus PCC7942 biotin 387  e−106gb|U04641|HSU04641 Human pyruvate carboxylase (PC) mRNA 387  e−106ref|NM_000920.1|PC| Homo sapiens pyruvate carboxylase (PC) 386  e−105gb|AE001090|AE001090 Archaeoglobus fulgidus section 17 383  e−104dbj|D84432|BACJH642 Bacillus subtilis DNA, 283 Kb region 382  e−104emb|Z99116|BSUB0013 Bacillus subtilis complete genome 382  e−104gb|AE000942|AE000942 Methanobacterium thermoautotrophicum 382  e−104gb|S72370|S72370 pyruvate carboxylase human, kidney 380  e−104dbj|D64001|SYCCPNC Synechocystis sp. PCC6803 complete 379  e−103gb|L14612|PSEACCBC Pseudomonas aeruginosa biotin carboxyl 376  e−103gb|U32778|U32778 Haemophilus influenzae Rd section 93 375  e−102emb|Z36087|SCYBR218C S. cerevisiae chromosome II 374  e−102gb|U35647|SCU35647 Saccharomyces cerevisiae pyruvate 374  e−102gb|J03889|YSCPCB Yeast (S. cerevisiae) pyruvate carboxylase 374  e−102gb|U90879|ATU90879 Arabidopsis thaliana biotin carboxylase 374  e−102emb|Z72584|SCYGL062W S. cerevisiae chromosome VII 374  e−102emb|X59890|SCPYC2G S. cerivisiae PYC2 gene for pyruvate 373  e−102gb|AE000749|AE000749 Aquifex aeolicus section 81 371  e−101gb|AE001286|AE001286 Chlamydia trachomatis section 13 370  e−101gb|AE001604|AE001604 Chlamydia pneumoniae section 20 369  e−100gb|AF007100|AF007100 Glycine max biotin carboxylase 368  e−100emb|Z95556|MTCY07A7 Mycobacterium tuberculosis H37Rv 367  e−100emb|Z19549|MTBCARBCP M. tuberculosis gene for biotin 367  e−100gb|AF068249|AF068249 Glycine max biotin carboxylase 366 1e−99gb|L38260|TOBBCSO Nicotiana tabacum acetyl CoA 363 7e−99gb|U36245|BSU36245 Bacillus subtilis biotin carboxyl 362 2e−98gb|AF097728|AF097728 Aspergillus terreus pyruvate 361 3e−98emb|AJ235272|RPXX03 Rickettsia prowazekii strain Madrid E 360 1e−97dbj|D83706|D83706 Bacillus stearothermophilus DNA 360 1e−97gb|AE000744|AE000744 Aquifex aeolicus section 76 358 3e−97emb|AL109846.1|SPBC17G9 S. pombe chromosome II 356 1e−96dbj|D78170|D78170 Yeast DNA for pyruvate carboxylase 353 1e−95gb|M79446|ECOFABG Escherichia coli biotin carboxylase gene 352 2e−95gb|M83198|ECOFABEGF Escherichia coli biotin carboxyl 352 2e−95gb|AE00404|AE000404 Escherichia coli K-12 MG1655 352 2e−95gb|U18997.1|ECOUW67 Escherichia coli K-12 chromosomal 352 2e−95gb|M80458|ECOACOAC E. coli biotin carboxylase and biotin 352 2e−95gb|U51439|REU51439 Rhizobium etli pyruvate carboxylase 351 5e−95emb|Y13917|BSY13917 Bacillus subtilis ppsE, yngL, yngK 348 3e−94emb|Z99113|BSUB0010 Bacillus subtilis complete genome 348 3e−94gb|AE001274.1|AE001274 Leishmania major chromosome 1 347 6e−94gb|AF042099|AF042099 Sulfolobus metallicus putative 346 1e−93emb|Z81052.1|CED2023 Caenorhabditis elegans cosmid D2023 162 3e−92emb|Z79700|MTCY10D7 Mycobacterium tuberculosis H37Rv 341 4e−92emb|Z99111|BSUB0008 Bacillus subtilis complete genome 340 1e−91gb|U12536|ATU12536 Arabidopsis thaliana 3-methylcrotonyl 338 4e−91emb|Y11106|PPPYC1 P. pastoris PYC1 gene 338 4e−91 gb|AE001529|AE001529Helicobacter pylori, strain J99 334 5e−90 gb|AE000553.1|AE000553Helicobacter pylori 26695 333 7e−90 emb|Y09548|CGPYC Corynebacteriumglutamicum pyc gene 333 1e−89 gb|AF038548|AF038548 Corynebacteriumglutamicum pyruvate 333 1e−89 ref|NM_000282.1|PCCA| Homo sapiensPropionyl Coenzyme 333 1e−89 gb|M22631|RATPCOA Rat alpha-propionyl CoAcarboxylase 332 2e−89 gb|U08469|GMU08469 Glycine max 3-methylcrotonylCoA 328 3e−88 emb|Z83018|MTCY349 Mycobacterium tuberculosis H37Rv 3184e−85 emb|AJ243652.1|PFL243652 Pseudomonas fluorescens uahA gene 3161e−84 emb|Z36077|SCYBR208C S. cerevisiae chromosome II 312 2e−83gb|M64926|YSCUAMD Yeast urea amidolyase (DUR1.2) gene 311 5e−83emb|Z97025|BSZ97025 Bacillus subtilis nprE, yla[A, B, C, D, E, F 3001e−79 emb|Z81074.1|CEF32B6 Caenorhabditis elegans cosmid F32B6 131 7e−78gb|U00024|MTU00024 Mycobacterium tuberculosis cosmid tbc2 284 7e−75gb|AD000009|MSGY2 Mycobacterium tuberculosis sequence 284 7e−75gb|U34393|GMU34393 Glycine max acetyl CoA carboxylase 259 2e−67gb|U49829|CELF27D9 Caenorhabditis elegans cosmid F27D9 186 4e−59emb|AJ010111.1|BCE010111 Bacillus cereus pycA, ctaA, ctaB 208 5e−52gb|U19183|ZMU19183 Zea mays acetyl-coenzyme A carboxylase 208 5e−52gb|U10187|TAU10187 Triticum aestivum Tam 107 206 2e−51gb|AF029895|AF029895 Triticum aestivum acetyl-coenzyme A 205 5e−51gb|J03808|RATACACA Rat acetyl-coenzyme A carboxylase mRNA 204 8e−51emb|X80045|OAACOAC O. aries mRNA for acetyl CoA carboxylase 203 1e−50emb|X68968|HSACOAC H. sapiens mRNA for acetyl CoA 203 2e−50emb|AJ132890.1|BTA132890 Bos taurus mRNA for acetyl 202 2e−50gb|J03541|CHKCOACA Chicken acetyl CoA carboxylase mRNA 202 3e−50dbj|D34630|ATHACCRNA Arabidopsis thaliana mRNA 199 2e−49gb|L25042|ALFACCASE Medicago sativa acetyl CoA carboxylase 198 5e−49emb|Z71631|SCYNR016C S. cerevisiae chromosome XIV 193 2e−47gb|M92156|YSCFAS3A Saccharomyces cerevisiae acetyl CoA 193 2e−47emb|Z49809|SC8261X S. cerevisiae chromosome XIII cosmid 8261 192 3e−47emb|Z22558|SCHFA1GN S. cerevisiae HFA1 gene 192 3e−47 dbj|D78165|D78165Saccharomyces cerevisiae DNA 192 3e−47 emb|Z46886|UMACCGEN U. maydis ACCgene for acetyl coa 190 1e−46 ref|NM_001093.1|ACACB| Homo sapiens acetylCoenzyme A 181 5e−44 Propionyl CoA carboxylase (accA1)gb|AF113603.1|AF113603 S. coelicolor putative 1101 0.0 (query sequence)gb|AF113604.1|AF113604 Streptomyces coelicolor putative 1090 0.0gb|AF126429.1|AF126429 Streptomyces venezuelae JadJ (jadJ) 967 0.0emb|Z92771|MTCY71 Mycobacterium tuberculosis H37Rv 758 0.0emb|X92557|SEPCCBBCP S. erythraea pccB, bcpA2, and orfX genes 753 0.0emb|X92556|SEHGTABCP S. erythraea hgtA, bcpA1, and orf122 753 0.0gb|U00012|U00012 Mycobacterium leprae cosmid B1308 745 0.0emb|X63470|MLBCCPG M. leprae gene for biotin carboxyl 742 0.0gb|U35023|CGU35023 Corynebacterium glutamicum thiosulfate 694 0.0gb|U24659|SVU24659 Streptomyces venezuelae glucose 596  e−169gb|AE000742|AE000742 Aquifex aeolicus section 74 417  e−115gb|U67563|U67563 Methanococcus jannaschii section 105 413  e−114gb|L36530|MQSPYRCARB Aedes aegypti pyruvate carboxylase 404  e−111gb|AF132152.1|AF132152 Drosophila melanogaster clone 400  e−110gb|L09192|MUSMPYR Mus musculus pyruvate carboxylase 397  e−109gb|U36585|RNU36585 Rattus norvegicus pyruvate carboxylase 395  e−108gb|U32314|RNU32314 Rattus norvegicus pyruvate carboxylase 395  e−108gb|L14862|ANAACCC Anabaena sp. (PCC 7120) 49.1 kDa biotin 394  e−108gb|U04641|HSU04641 Human pyruvate carboxylase (PC) mRNA 391  e−107gb|U59234|SPU59234 Synechococcus PCC7942 biotin carboxylase 391  e−107ref|NM_000920.1|PC| Homo sapiens pyruvate carboxylase (PC) 390  e−107gb|AE001090|AE001090 Archaeoglobus fulgidus section 17 389  e−106gb|AE000942|AE000942 Methanobacterium thermoautotrophicum 386  e−105gb|S72370|S72370 pyruvate carboxylase human, kidney 384  e−105dbj|D84432|BACJH642 Bacillus subtilis DNA, 283 Kb region 383  e−105emb|Z99116|BSUB0013 Bacillus subtilis complete genome 383  e−105dbj|D64001|SYCCPNC Synechocystis sp. PCC6803 383  e−104gb|U35647|SCU35647 Saccharomyces cerevisiae pyruvate 382  e−104emb|Z36087|SCYBR218C S. cerevisiae chromosome II 382  e−104emb|Z72584|SCYGL062W S. cerevisiae chromosome VII 381  e−104gb|J03889|YSCPCB Yeast (S. cerevisiae) pyruvate carboxylase 381  e−104gb|L14612|PSEACCBC Pseudomonas aeruginosa biotin carboxyl 381  e−104emb|X59890|SCPYC2G S. cerivisiae PYC2 gene for pyruvate 381  e−104gb|U32778|U32778 Haemophilus influenzae Rd section 93 380  e−104gb|U90879|ATU90879 Arabidopsis thaliana biotin carboxylase 377  e−103gb|AE000749|AE000749 Aquifex aeolicus section 81 of 109 377  e−103gb|AE001286|AE001286 Chlamydia trachomatis section 13 375  e−102gb|AE001604|AE001604 Chlamydia pneumoniae section 20 374  e−102gb|AF007100|AF007100 Glycine max biotin carboxylase 372  e−101emb|Z95556|MTCY07A7 Mycobacterium tuberculosis H37Rv 369  e−100emb|Z19549|MTBCARBCP M. tuberculosis gene for biotin 369  e−100gb|AF068249|AF068249 Glycine max biotin carboxylase 369  e−100gb|L38260|TOBBCSO Nicotiana tabacum acetyl CoA 367  e−100gb|AF097728|AF097728 Aspergillus terreus pyruvate 366 1e−99gb|AE000744|AE000744 Aquifex aeolicus section 76 of 109 364 4e−99dbj|D83706|D83706 Bacillus stearothermophilus DNA 363 7e−99gb|U36245|BSU36245 Bacillus subtilis biotin carboxyl 363 7e−99emb|AL109846.1|SPBC17G9 S. pombe chromosome II 362 2e−98emb|AJ235272|RPXX03 Rickettsia prowazekii strain Madrid E 361 3e−98dbj|D78170|D78170 Yeast DNA for pyruvate carboxylase 359 2e−97gb|M80458|ECOACOAC E. coli biotin carboxylase and biotin 358 3e−97gb|M79446|ECOFABG Escherichia coli biotin carboxylase gene 358 3e−97gb|M83198|ECOFABEGF Escherichia coli biotin carboxyl 358 3e−97gb|AE000404|AE000404 Escherichia coli K-12 MG1655 358 3e−97gb|U18997.1|ECOUW67 Escherichia coli K-12 chromosomal 358 3e−97gb|U51439|REU51439 Rhizobium etli pyruvate carboxylase 355 3e−96emb|Y13917|BSY13917 Bacillus subtilis ppsE, yngL, yngK, 354 4e−96emb|Z99113|BSUB0010 Bacillus subtilis complete genome 354 4e−96gb|AE001274.1|AE001274 Leishmania major chromosome 1 351 3e−95gb|AF042099|AF042099 Sulfolobus metallicus putative 350 9e−95emb|Z79700|MTCY10D7 Mycobacterium tuberculosis H37Rv 347 6e−94emb|Z81052.1|CED2023 Caenorhabditis elegans cosmid D2023 168 1e−93emb|Y11106|PPPYC1 P. pastoris PYC1 gene 345 2e−93 emb|Z99111|BSUB0008Bacillus subtilis complete genome 343 8e−93 ref|NM_000282.1|PCCA| Homosapiens Propionyl Coenzyme 340 6e−92 gb|M22631|RATPCOA Ratalpha-propionyl CoA carboxylase 340 1e−91 gb|U12536|ATU12536 Arabidopsisthaliana 3-methylcrotonyl 339 2e−9 emb|Y09548|CGPYC Corynebacteriumglutamicum pyc gene 338 4e−91 gb|AF038548|AF038548 Corynebacteriumglutamicum pyruvate 338 4e−91 gb|AE001529|AE001529 Helicobacter pylori,strain J99 337 8e−91 gb|AE000553.1|AE000553 Helicobacter pylori 26695336 1e−90 gb|U08469|GMU08469 Glycine max 3-methylcrotonyl CoA 329 2e−88emb|AJ243652.1|PFL243652 Pseudomonas fluorescens uahA gene 323 1e−86emb|Z83018|MTCY349 Mycobacterium tuberculosis H37Rv 321 3e−86emb|Z36077|SCYBR208C S. cerevisiae chromosome II 314 5e−84gb|M64926|YSCUAMD Yeast urea amidolyase (DUR1.2) gene 312 2e−83emb|Z97025|BSZ97025 Bacillus subtilis nprE, yla[A, B, C, D, E, 303 1e−80emb|Z81074.1|CEF32B6 Caenorhabditis elegans cosmid F32B6 130 1e−78gb|U00024|MTU00024 Mycobacterium tuberculosis cosmid tbc2 287 6e−76gb|AD000009|MSGY2 Mycobacterium tuberculosis sequence 287 6e−76gb|U34393|GMU34393 Glycine max acetyl CoA carboxylase 262 3e−68gb|U49829|CELF27D9 Caenorhabditis elegans cosmid F27D9 190 2e−61gb|U10187|TAU10187 Triticum aestivum Tam 107 213 2e−53gb|U19183|ZMU19183 Zea mays acetyl-coenzyme A carboxylase 212 3e−53emb|AJ010111.1|BCE010111 Bacillus cereus pycA, ctaA, ctaB 212 4e−53gb|AF029895|AF029895 Triticum aestivum acetyl-coenzyme 209 2e−52gb|J03808|RATACACA Rat acetyl-coenzyme A carboxylase 205 4e−51emb|X80045|OAACOAC O. aries mRNA for acetyl CoA 205 5e−51emb|X68968|HSACOAC H. sapiens mRNA for acetyl CoA 204 8e−51dbj|D34630|ATHACCRNA Arabidopsis thaliana mRNA 203 1e−50emb|AJ132890.1|BTA132890 Bos taurus mRNA for acetyl CoA 203 1e−50gb|J03541|CHKCOACA Chicken acetyl CoA carboxylase mRNA 203 1e−50gb|L25042|ALFACCASE Medicago sativa acetyl CoA carboxylase 202 2e−50emb|Z71631|SCYNR016C S. cerevisiae chromosome XIV 196 1e−48gb|M92156|YSCFAS3A Saccharomyces cerevisiae acetyl CoA 196 1e−48emb|Z49809|SC8261X S. cerevisiae chromosome XIII cosmid 8261 195 4e−48emb|Z22558|SCHFA1GN S. cerevisiae HFA1 gene 195 4e−48 dbj|D78165|D78165Saccharomyces cerevisiae DNA 195 4e−48 emb|Z46886|UMACCGEN U. maydis ACCgene for acetyl coa 188 5e−46 gb|L20784|CCXACOAC Cyclotella crypticaacetyl CoA 182 2e−44

Those of skill in the art will recognize that, due to the degeneratenature of the genetic code, a variety of DNA compounds differing intheir nucleotide sequences can be used to encode a given amino acidsequence of the invention. The native DNA sequence encoding thebiosynthetic enzymes in the tables above are referenced herein merely toillustrate a preferred embodiment of the invention, and the inventionincludes DNA compounds of any sequence that encode the amino acidsequences of the polypeptides and proteins of the enzymes utilized inthe methods of the invention. In similar fashion, a polypeptide cantypically tolerate one or more amino acid substitutions, deletions, andinsertions in its amino acid sequence without loss or significant lossof a desired activity. The present invention includes such polypeptideswith alternate amino acid sequences, and the amino acid sequencesencoded by the DNA sequences shown herein merely illustrate preferredembodiments of the invention.

Thus, in an especially preferred embodiment, the present inventionprovides DNA molecules in the form of recombinant DNA expression vectorsor plasmids, as described in more detail below, that encode one or moreprecursor biosynthetic enzymes. Generally, such vectors can eitherreplicate in the cytoplasm of the host cell or integrate into thechromosomal DNA of the host cell. In either case, the vector can be astable vector (i.e., the vector remains present over many celldivisions, even if only with selective pressure) or a transient vector(i.e., the vector is gradually lost by host cells with increasingnumbers of cell divisions). The invention provides DNA molecules inisolated (i.e., not pure, but existing in a preparation in an abundanceand/or concentration not found in nature) and purified (i.e.,substantially free of contaminating materials or substantially free ofmaterials with which the corresponding DNA would be found in nature)form.

In one important embodiment, the invention provides methods for theheterologous expression of one or more of the biosynthetic genesinvolved in S-methylmalonyl CoA biosynthesis and recombinant DNAexpression vectors useful in the method. Thus, included within the scopeof the invention are recombinant expression vectors that include suchnucleic acids. The term expression vector refers to a nucleic acid thatcan be introduced into a host cell or cell-free transcription andtranslation system. An expression vector can be maintained permanentlyor transiently in a cell, whether as part of the chromosomal or otherDNA in the cell or in any cellular compartment, such as a replicatingvector in the cytoplasm. An expression vector also comprises a promoterthat drives expression of an RNA, which typically is translated into apolypeptide in the cell or cell extract. For efficient translation ofRNA into protein, the expression vector also typically contains aribosome-binding site sequence positioned upstream of the start codon ofthe coding sequence of the gene to be expressed. Other elements, such asenhancers, secretion signal sequences, transcription terminationsequences, and one or more marker genes by which host cells containingthe vector can be identified and/or selected, may also be present in anexpression vector. Selectable markers, i.e., genes that conferantibiotic resistance or sensitivity, are preferred and confer aselectable phenotype on transformed cells when the cells are grown in anappropriate selective medium.

The various components of an expression vector can vary widely,depending on the intended use of the vector and the host cell(s) inwhich the vector is intended to replicate or drive expression.Expression vector components suitable for the expression of genes andmaintenance of vectors in E. coli, yeast, Streptomyces, and othercommonly used cells are widely known and commercially available. Forexample, suitable promoters for inclusion in the expression vectors ofthe invention include those that function in eucaryotic or procaryotichost cells. Promoters can comprise regulatory sequences that allow forregulation of expression relative to the growth of the host cell or thatcause the expression of a gene to be turned on or off in response to achemical or physical stimulus. For E. coli and certain other bacterialhost cells, promoters derived from genes for biosynthetic enzymes,antibiotic-resistance conferring enzymes, and phage proteins can be usedand include, for example, the galactose, lactose (lac), maltose,tryptophan (trp), beta-lactamase (bla), bacteriophage lambda PL, and T5promoters. In addition, synthetic promoters, such as the tac promoter(U.S. Pat. No. 4,551,433), can also be used. For E. coli expressionvectors, it is useful to include an E. coli origin of replication, suchas from pUC, p1P, p1I, and pBR.

Thus, recombinant expression vectors contain at least one expressionsystem, which, in turn, is composed of at least a portion of PKS and/orother biosynthetic gene coding sequences operably linked to a promoterand optionally termination sequences that operate to effect expressionof the coding sequence in compatible host cells. The host cells aremodified by transformation with the recombinant DNA expression vectorsof the invention to contain the expression system sequences either asextrachromosomal elements or integrated into the chromosome. Theresulting host cells of the invention are useful in methods to producePKS enzymes as well as polyketides and antibiotics and other usefulcompounds derived therefrom.

Preferred host cells for purposes of selecting vector components forexpression vectors of the present invention include fungal host cellssuch as yeast and procaryotic host cells such as E. coli, but mammalianhost cells can also be used. In hosts such as yeasts, plants, ormammalian cells that ordinarily do not produce polyketides, it may benecessary to provide, also typically by recombinant means, suitableholo-ACP synthases to convert the recombinantly produced PKS tofunctionality. Provision of such enzymes is described, for example, inPCT publication Nos. WO 97/13845 and 98/27203, each of which isincorporated herein by reference.

The recombinant host cells of the invention can express all of thepolyketide biosynthetic genes or only a subset of the same. For example,if only the genes for a PKS are expressed in a host cell that otherwisedoes not produce polyketide modifying enzymes (such as hydroxylation,epoxidation, or glycosylation enzymes) that can act on the polyketideproduced, then the host cell produces unmodified polyketides, calledmacrolide aglycones. Such macrolide aglycones can be hydroxylated andglycosylated by adding them to the fermentation of a strain such as, forexample, Streptomyces antibioticus or Saccharopolyspora erythraea, thatcontains the requisite modification enzymes.

There are a wide variety of diverse organisms that can modify macrolideaglycones to provide compounds with, or that can be readily modified tohave, useful activities. For example, Saccharopolyspora eythraea canconvert 6-dEB to a variety of useful compounds. The erythronolide 6-dEBis converted by the eryF gene product to erythronolide B, which is, inturn, glycosylated by the eryB gene product to obtain3-O-mycarosylerythronolide B, which contains L-mycarose at C-3. Theenzyme eryC gene product then converts this compound to erythromycin Dby glycosylation with D-desosamine at C-5. Erythromycin D, therefore,differs from 6-dEB through glycosylation and by the addition of ahydroxyl group at C-6. Erythromycin D can be converted to erythromycin Bin a reaction catalyzed by the eryG gene product by methylating theL-mycarose residue at C-3. Erythromycin D is converted to erythromycin Cby the addition of a hydroxyl group at C-12 in a reaction catalyzed bythe eryK gene product. Erythromycin A is obtained from erythromycin C bymethylation of the mycarose residue in a reaction catalyzed by the eryGgene product. The unmodified polyketides provided by the presentinvention, such as, for example, 6-dEB produced in E. coli, can beprovided to cultures of S. erythraea and converted to the correspondingderivatives of erythromycins A, B, C, and D in accordance with theprocedure provided in the examples below. To ensure that only thedesired compound is produced, one can use an S. erythraea eryA mutantthat is unable to produce 6-dEB but can still carry out the desiredconversions (Weber et al., 1985, J. Bacteriol. 164(1): 425-433). Also,one can employ other mutant strains, such as eryB, eryC, eryG, and/oreryK mutants, or mutant strains having mutations in multiple genes, toaccumulate a preferred compound. The conversion can also be carried outin large fermentors for commercial production.

Moreover, there are other useful organisms that can be employed tohydroxylate and/or glycosylate the compounds of the invention. Asdescribed above, the organisms can be mutants unable to produce thepolyketide normally produced in that organism, the fermentation can becarried out on plates or in large fermentors, and the compounds producedcan be chemically altered after fermentation. Thus, Streptomycesvenezuelae, which produces picromycin, contains enzymes that cantransfer a desosaminyl group to the C-5 hydroxyl and a hydroxyl group tothe C-12 position. In addition, S. venezuelae contains a glucosylationactivity that glucosylates the 2′-hydroxyl group of the desosaminesugar. This latter modification reduces antibiotic activity, but theglucosyl residue is removed by enzymatic action prior to release of thepolyketide from the cell. Another organism, S. narbonensis, contains thesame modification enzymes as S. venezuelae, except the C-12 hydroxylase.Thus, the present invention provides the compounds produced byhydroxylation and glycosylation of the macrolide aglycones of theinvention by action of the enzymes endogenous to S. narbonensis and S.venezuelae.

Other organisms suitable for making compounds of the invention includeMicromonospora megalomicea, Streptomyces antibioticus, S. fradiae, andS. thermotolerans. M. megalomicea glycosylates the C-3 hydroxyl withmycarose, the C-5 hydroxyl with desosamine, and the C-6 hydroxyl withmegosamine, and hydroxylates the C-6 position. S. antibioticus producesoleandomycin and contains enzymes that hydroxylate the C-6 and C-12positions, glycosylate the C-3 hydroxyl with oleandrose and the C-5hydroxyl with desosamine, and form an epoxide at C-8-C-8a. S. fradiaecontains enzymes that glycosylate the C-5 hydroxyl with mycaminose andthen the 4′-hydroxyl of mycaminose with mycarose, forming adisaccharide. S. thermotolerans contains the same activities as S.fradiae, as well as acylation activities. Thus, the present inventionprovides the compounds produced by hydroxylation and glycosylation ofthe macrolide aglycones of the invention by action of the enzymesendogenous to M. megalomicea, S. antibioticus, S. fradiae, and S.thermotolerans.

The present invention also provides methods and genetic constructs forproducing the glycosylated and/or hydroxylated compounds of theinvention directly in the host cell of interest. Thus, the genes thatencode polyketide modification enzymes can be included in the host cellsof the invention. Lack of adequate resistance to a polyketide can beovercome by providing the host cell with an MLS resistance gene (ermEand mgt/lrm, for example), which confer resistance to several14-membered macrolides (see Cundliffe, 1989, Annu. Rev. Microbiol.43:207-33; Jenkins and Cundliffe, 1991, Gene 108:55-62; and Cundliffe,1992, Gene, 115:75-84, each of which is incorporated herein byreference).

The recombinant host cells of the invention can be used to producepolyketides (both macrolide aglycones and their modified derivatives)that are naturally occurring or produced by recombinant DNA technology.In one important embodiment, the recombinant host cells of the inventionare used to produce hybrid PKS enzymes. For purposes of the invention, ahybrid PKS is a recombinant PKS that comprises all or part of one ormore extender modules, loading module, and/or thioesterase/cyclasedomain of a first PKS and all or part of one or more extender modules,loading module, and/or thioesterase/cyclase domain of a second PKS.

Those of skill in the art will recognize that all or part of either thefirst or second PKS in a hybrid PKS of the invention need not beisolated from a naturally occurring source. For example, only a smallportion of an AT domain determines its specificity. See PCT patentapplication No. WO US99/15047 (PCT publication No. WO 00/08138), and Lauet al., infra, incorporated herein by reference. The state of the art inDNA synthesis allows the artisan to construct de novo DNA compounds ofsize sufficient to construct a useful portion of a PKS module or domain.Thus, the desired derivative coding sequences can be synthesized usingstandard solid phase synthesis methods such as those described by Jayeet al., 1984, J. Biol. Chem. 259: 6331, and instruments for automatedsynthesis are available commercially from, for example, AppliedBiosystems, Inc. For purposes of the invention, such synthetic DNAcompounds are deemed to be a portion of a PKS.

A hybrid PKS for purposes of the present invention can result not only:

(i) from fusions of heterologous domain (where heterologous means thedomains in a module are derived from at least two different naturallyoccurring modules) coding sequences to produce a hybrid module codingsequence contained in a PKS gene whose product is incorporated into aPKS,

but also:

(ii) from fusions of heterologous module (where heterologous modulemeans two modules are adjacent to one another that are not adjacent toone another in naturally occurring PKS enzymes) coding sequences toproduce a hybrid coding sequence contained in a PKS gene whose productis incorporated into a PKS,

(iii) from expression of one or more PKS genes from a first PKS genecluster with one or more PKS genes from a second PKS gene cluster, and

(iv) from combinations of the foregoing.

Various hybrid PKSs of the invention illustrating these variousalternatives are described herein.

Recombinant methods for manipulating modular PKS genes to make hybridPKS enzymes are described in U.S. Pat. Nos. 5,672,491; 5,843,718;5,830,750; and 5,712,146; and in PCT publication Nos. 98/49315 and97/02358, each of which is incorporated herein by reference. A number ofgenetic engineering strategies have been used with DEBS to demonstratethat the structures of polyketides can be manipulated to produce novelnatural products, primarily analogs of the erythromycins (see the patentpublications referenced supra and Hutchinson, 1998, Curr Opin Microbiol.1:319-329, and Baltz, 1998, Trends Microbiol. 6:76-83, incorporatedherein by reference).

These techniques include: (i) deletion or insertion of modules tocontrol chain length, (ii) inactivation of reduction/dehydration domainsto bypass beta-carbon processing steps, (iii) substitution of AT domainsto alter starter and extender units, (iv) addition ofreduction/dehydration domains to introduce catalytic activities, and (v)substitution of ketoreductase KR domains to control hydroxylstereochemistry. In addition, engineered blocked mutants of DEBS havebeen used for precursor directed biosynthesis of analogs thatincorporate synthetically derived starter units. For example, more than100 novel polyketides were produced by engineering single andcombinatorial changes in multiple modules of DEBS. Hybrid PKS enzymesbased on DEBS with up to three catalytic domain substitutions wereconstructed by cassette mutagenesis, in which various DEBS domains werereplaced with domains from the rapamycin PKS (see Schweke et al., 1995,Proc. Nat. Acad. Sci. USA 92, 7839-7843, incorporated herein byreference) or one more of the DEBS KR domains was deleted. Functionalsingle domain replacements or deletions were combined to generate DEBSenzymes with double and triple catalytic domain substitutions (seeMcDaniel et al., 1999, Proc. Nat. Acad. Sci. USA 96, 1846-1851,incorporated herein by reference).

Methods for generating libraries of polyketides have been greatlyimproved by cloning PKS genes as a set of three or more mutuallyselectable plasmids, each carrying a different wild-type or mutant PKSgene, then introducing all possible combinations of the plasmids withwild-type, mutant, and hybrid PKS coding sequences into the same host(see U.S. patent application Ser. No. 60/129,731, filed 16 Apr. 1999,and PCT Pub. No. WO 98/27203, each of which is incorporated herein byreference). This method can also incorporate the use of a KS1° mutant,which by mutational biosynthesis can produce polyketides made fromdiketide starter units (see Jacobsen et al., 1997, Science 277, 367-369,incorporated herein by reference), as well as the use of a truncatedgene that leads to 12-membered macrolides or an elongated gene thatleads to 16-membered ketolides. Moreover, by utilizing in addition oneor more vectors that encode glycosyl biosynthesis and transfer genes,such as those of the present invention for megosamine, desosamine,oleandrose, cladinose, and/or mycarose (in any combination), a largecollection of glycosylated polyketides can be prepared.

The following table lists references describing illustrative PKS genesand corresponding enzymes that can be utilized in the construction ofthe recombinant hybrid PKSs and the corresponding DNA compounds thatencode them. Also presented are various references describing tailoringenzymes and corresponding genes that can be employed in accordance withthe methods of the invention.

Avermectin

-   U.S. Pat. No. 5,252,474 to Merck.-   MacNeil et al., 1993, Industrial Microorganisms: Basic and Applied    Molecular Genetics, Baltz, Hegeman, & Skatrud, eds. (ASM), pp.    245-256, A Comparison of the Genes Encoding the Polyketide Synthases    for Avermectin, Erythromycin, and Nemadectin.

MacNeil et al., 1992, Gene 115: 119-125, Complex Organization of theStreptomyces avermitilis genes encoding the avermectin polyketidesynthase.

Candicidin (FR008)

-   Hu et al., 1994, Mol. Microbiol. 14: 163-172.

Epothilone

-   PCT Pat. Pub. No. WO 00/031247 to Kosan.

Erythromycin

-   PCT Pub. No. 93/13663 to Abbott.-   U.S. Pat. No. 5,824,513 to Abbott.-   Donadio et al., 1991, Science 252:675-9.-   Cortes et al., 8 Nov. 1990, Nature 348:176-8, An unusually large    multifunctional polypeptide in the erythromycin producing polyketide    synthase of Saccharopolyspora erythraea.

Glycosylation Enzymes

-   PCT Pat. App. Pub. No. 97/23630 to Abbott.

FK-506

-   Motamedi et al., 1998, The biosynthetic gene cluster for the    macrolactone ring of the immunosuppressant FK506, Eur. J. biochem.    256: 528-534.-   Motamedi et al., 1997, Structural organization of a multifunctional    polyketide synthase involved in the biosynthesis of the macrolide    immunosuppressant FK506, Eur. J. Biochem. 244: 74-80.

Methyltransferase

-   U.S. Pat. No. 5,264,355, issued 23 Nov. 1993, Methylating enzyme    from Streptomyces MA6858. 31-O-desmethyl-FK506 methyltransferase.-   Motamedi et al., 1996, Characterization of methyltransferase and    hydroxylase genes involved in the biosynthesis of the    immunosuppressants FK506 and FK520, J. Bacteriol. 178: 5243-5248.

FK-520

-   PCT Pat. Pub. No. WO 00/020601 to Kosan. See also Nielsen et al.,    1991, Biochem. 30:5789-96 (enzymology of pipecolate incorporation).

Lovastatin

-   U.S. Pat. No. 5,744,350 to Merck.

Narbomycin (and Picromycin)

-   PCT Pat. Pub. No. WO 99/61599 to Kosan.

Nemadectin

-   MacNeil et al., 1993, supra.

Niddamycin

Kakavas et al., 1997, Identification and characterization of theniddamycin polyketide synthase genes from Streptomyces caelestis, J.Bacteriol. 179: 7515-7522.

Oleandomycin

-   Swan et al., 1994, Characterisation of a Streptomyces antibioticus    gene encoding a type I polyketide synthase which has an unusual    coding sequence, Mol. Gen. Genet. 242: 358-362.-   PCT Pat. Pub. No. WO 00/026349 to Kosan.-   Olano et al., 1998, Analysis of a Streptomyces antibioticus    chromosomal region involved in oleandomycin biosynthesis, which    encodes two glycosyltransferases responsible for glycosylation of    the macrolactone ring, Mol. Gen. Genet. 259(3): 299-308.

Platenolide

-   EP Pat. App. Pub. No. 791,656 to Lilly.

Rapamycin

-   Schwecke et al., August 1995, The biosynthetic gene cluster for the    polyketide rapamycin, Proc. Natl. Acad. Sci. USA 92:7839-7843.-   Aparicio et al., 1996, Organization of the biosynthetic gene cluster    for rapamycin in Streptomyces hygroscopicus: analysis of the    enzymatic domains in the modular polyketide synthase, Gene 169:    9-16.

Rifamycin

August et al., 13 Feb. 1998, Biosynthesis of the ansamycin antibioticrifamycin: deductions from the molecular analysis of the rifbiosynthetic gene cluster of Amycolatopsis mediterranei S669, Chemistry& Biology, 5(2): 69-79.

Soraphen

-   U.S. Pat. No. 5,716,849 to Novartis.-   Schupp et al., 1995, J. Bacteriology 177: 3673-3679. A Sorangium    cellulosum (Myxobacterium) Gene Cluster for the Biosynthesis of the    Macrolide Antibiotic Soraphen A: Cloning, Characterization, and    Homology to Polyketide Synthase Genes from Actinomycetes.

Spiramycin

-   U.S. Pat. No. 5,098,837 to Lilly.

Activator Gene

-   U.S. Pat. No. 5,514,544 to Lilly.

Tylosin

-   EP Pub. No. 791,655 to Lilly.-   Kuhstoss et al., 1996, Gene 183:231-6., Production of a novel    polyketide through the construction of a hybrid polyketide synthase.-   U.S. Pat. No. 5,876,991 to Lilly.

Tailoring Enzymes

-   Merson-Davies and Cundliffe, 1994, Mol. Microbiol. 13: 349-355.    Analysis of five tylosin biosynthetic genes from the tylBA region of    the Streptomyces fradiae genome.

As the above Table illustrates, there are a wide variety of PKS genesthat serve as readily available sources of DNA and sequence informationfor use in constructing the hybrid PKS-encoding DNA compounds of theinvention.

In constructing hybrid PKSs, certain general methods may be helpful. Forexample, it is often beneficial to retain the framework of the module tobe altered to make the hybrid PKS. Thus, if one desires to add DH and ERfunctionalities to a module, it is often preferred to replace the KRdomain of the original module with a KR, DH, and ER domain-containingsegment from another module, instead of merely inserting DH and ERdomains. One can alter the stereochemical specificity of a module byreplacement of the KS domain with a KS domain from a module thatspecifies a different stereochemistry. See Lau et al., 1999, “Dissectingthe role of acyltransferase domains of modular polyketide synthases inthe choice and stereochemical fate of extender units” Biochemistry38(5): 1643-1651, incorporated herein by reference. One can alter thespecificity of an AT domain by changing only a small segment of thedomain. See Lau et al., supra. One can also take advantage of knownlinker regions in PKS proteins to link modules from two different PKSsto create a hybrid PKS. See Gokhale et al., 16 Apr. 1999, Dissecting andExploiting Intermodular Communication in Polyketide Synthases”, Science284: 482-485, incorporated herein by reference.

The hybrid PKS-encoding DNA compounds can be and often are hybrids ofmore than two PKS genes. Even where only two genes are used, there areoften two or more modules in the hybrid gene in which all or part of themodule is derived from a second (or third) PKS gene.

The invention also provides libraries of PKS genes, PKS proteins, andultimately, of polyketides, that are constructed by generatingmodifications in a PKS so that the protein complexes produced havealtered activities in one or more respects and thus produce polyketidesother than the natural product of the PKS. Novel polyketides may thus beprepared, or polyketides in general prepared more readily, using thismethod. By providing a large number of different genes or gene clustersderived from a naturally occurring PKS gene cluster, each of which hasbeen modified in a different way from the native cluster, an effectivelycombinatorial library of polyketides can be produced as a result of themultiple variations in these activities. As will be further describedbelow, the metes and bounds of this embodiment of the invention can bedescribed on the polyketide, protein, and the encoding nucleotidesequence levels.

There are at least five degrees of freedom for constructing a hybrid PKSin terms of the polyketide that will be produced. First, the polyketidechain length is determined by the number of extender modules in the PKS,and the present invention includes hybrid PKSs that contain 6, as wellsas fewer or more than 6, extender modules. Second, the nature of thecarbon skeleton of the PKS is determined by the specificities of theacyl transferases that determine the nature of the extender units ateach position, e.g., malonyl, methylmalonyl, ethylmalonyl, or othersubstituted malonyl. Third, the loading module specificity also has aneffect on the resulting carbon skeleton of the polyketide. The loadingmodule may use a different starter unit, such as acetyl, butyryl, andthe like. As noted above, another method for varying loading modulespecificity involves inactivating the KS activity in extender module 1(KS1) and providing alternative substrates, called diketides, that arechemically synthesized analogs of extender module 1 diketide products,for extender module 2. This approach was illustrated in PCT publicationNos. 97/02358 and 99/03986, incorporated herein by reference, whereinthe KS1 activity was inactivated through mutation. Fourth, the oxidationstate at various positions of the polyketide will be determined by thedehydratase and reductase portions of the modules. This will determinethe presence and location of ketone and alcohol moieties and C—C doublebonds or C—C single bonds in the polyketide. Finally, thestereochemistry of the resulting polyketide is a function of threeaspects of the synthase. The first aspect is related to the AT/KSspecificity associated with substituted malonyls as extender units,which affects stereochemistry only when the reductive cycle is missingor when it contains only a ketoreductase, as the dehydratase wouldabolish chirality. Second, the specificity of the ketoreductase maydetermine the chirality of any beta-OH. Finally, the enoylreductasespecificity for substituted malonyls as extender units may influence thestereochemistry when there is a complete KR/DH/ER available.

Thus, the modular PKS systems generally permit a wide range ofpolyketides to be synthesized. As compared to the aromatic PKS systems,the modular PKS systems accept a wider range of starter units, includingaliphatic monomers (acetyl, propionyl, butyryl, isovaleryl, etc.),aromatics (aminohydroxybenzoyl), alicyclics (cyclohexanoyl), andheterocyclics (thiazolyl). Certain modular PKSs have relaxed specificityfor their starter units (Kao et al., 1994, Science, supra). Modular PKSsalso exhibit considerable variety with regard to the choice of extenderunits in each condensation cycle. The degree of beta-ketoreductionfollowing a condensation reaction can be altered by genetic manipulation(Donadio et al., 1991, Science, supra; Donadio et al., 1993, Proc. Natl.Acad. Sci. USA 90: 7119-7123). Likewise, the size of the polyketideproduct can be varied by designing mutants with the appropriate numberof modules (Kao et al., 1994, J. Am. Chem. Soc. 116:11612-11613).Lastly, modular PKS enzymes are particularly well known for generatingan impressive range of asymmetric centers in their products in a highlycontrolled manner. The polyketides, antibiotics, and other compoundsproduced by the methods of the invention are typically singlestereoisomeric forms. Although the compounds of the invention can occuras mixtures of stereoisomers, it may be beneficial in some instances togenerate individual stereoisomers. Thus, the combinatorial potentialwithin modular PKS pathways based on any naturally occurring modular PKSscaffold is virtually unlimited.

While hybrid PKSs are most often produced by “mixing and matching”portions of PKS coding sequences, mutations in DNA encoding a PKS canalso be used to introduce, alter, or delete an activity in the encodedpolypeptide. Mutations can be made to the native sequences usingconventional techniques. The substrates for mutation can be an entirecluster of genes or only one or two of them; the substrate for mutationmay also be portions of one or more of these genes. Techniques formutation include preparing synthetic oligonucleotides including themutations and inserting the mutated sequence into the gene encoding aPKS subunit using restriction endonuclease digestion. See, e.g., Kunkel,1985, Proc. Natl. Acad. Sci. USA 82: 448; Geisselsoder et al., 1987,BioTechniques 5:786. Alternatively, the mutations can be effected usinga mismatched primer (generally 10-20 nucleotides in length) thathybridizes to the native nucleotide sequence, at a temperature below themelting temperature of the mismatched duplex. The primer can be madespecific by keeping primer length and base composition within relativelynarrow limits and by keeping the mutant base centrally located. SeeZoller and Smith, 1983, Methods Enzymol. 100:468. Primer extension iseffected using DNA polymerase, the product cloned, and clones containingthe mutated DNA, derived by segregation of the primer extended strand,selected. Identification can be accomplished using the mutant primer asa hybridization probe. The technique is also applicable for generatingmultiple point mutations. See, e.g., Dalbie-McFarland et al., 1982,Proc. Natl. Acad. Sci. USA 79: 6409. PCR mutagenesis can also be used toeffect the desired mutations.

Random mutagenesis of selected portions of the nucleotide sequencesencoding enzymatic activities can also be accomplished by severaldifferent techniques known in the art, e.g., by inserting anoligonucleotide linker randomly into a plasmid, by irradiation withX-rays or ultraviolet light, by incorporating incorrect nucleotidesduring in vitro DNA synthesis, by error-prone PCR mutagenesis, bypreparing synthetic mutants, or by damaging plasmid DNA in vitro withchemicals. Chemical mutagens include, for example, sodium bisulfite,nitrous acid, nitrosoguanidine, hydroxylamine, agents which damage orremove bases thereby preventing normal base-pairing such as hydrazine orformic acid, analogues of nucleotide precursors such as 5-bromouracil,2-aminopurine, or acridine intercalating agents such as proflavine,acriflavine, quinacrine, and the like. Generally, plasmid DNA or DNAfragments are treated with chemical mutagens, transformed into E. coliand propagated as a pool or library of mutant plasmids.

In constructing a hybrid PKS of the invention, regions encodingenzymatic activity, i.e., regions encoding corresponding activities fromdifferent PKS synthases or from different locations in the same PKS, canbe recovered, for example, using PCR techniques with appropriateprimers. By “corresponding” activity encoding regions is meant thoseregions encoding the same general type of activity. For example, a KRactivity encoded at one location of a gene cluster “corresponds” to a KRencoding activity in another location in the gene cluster or in adifferent gene cluster. Similarly, a complete reductase cycle could beconsidered corresponding. For example, KR/DH/ER can correspond to a KRalone.

If replacement of a particular target region in a host PKS is to bemade, this replacement can be conducted in vitro using suitablerestriction enzymes. The replacement can also be effected in vivo usingrecombinant techniques involving homologous sequences framing thereplacement gene in a donor plasmid and a receptor region in a recipientplasmid. Such systems, advantageously involving plasmids of differingtemperature sensitivities are described, for example, in PCT publicationNo. WO 96/40968, incorporated herein by reference. The vectors used toperform the various operations to replace the enzymatic activity in thehost PKS genes or to support mutations in these regions of the host PKSgenes can be chosen to contain control sequences operably linked to theresulting coding sequences in a manner such that expression of thecoding sequences can be effected in an appropriate host.

However, simple cloning vectors may be used as well. If the cloningvectors employed to obtain PKS genes encoding derived PKS lack controlsequences for expression operably linked to the encoding nucleotidesequences, the nucleotide sequences are inserted into appropriateexpression vectors. This need not be done individually, but a pool ofisolated encoding nucleotide sequences can be inserted into expressionvectors, the resulting vectors transformed or transfected into hostcells, and the resulting cells plated out into individual colonies. Theinvention provides a variety of recombinant DNA compounds in which thevarious coding sequences for the domains and modules of the PKS areflanked by non-naturally occurring restriction enzyme recognition sites.

The various PKS nucleotide sequences can be cloned into one or morerecombinant vectors as individual cassettes, with separate controlelements, or under the control of, e.g., a single promoter. The PKSsubunit encoding regions can include flanking restriction sites to allowfor the easy deletion and insertion of other PKS subunit encodingsequences so that hybrid PKSs can be generated. The design of suchunique restriction sites is known to those of skill in the art and canbe accomplished using the techniques described above, such assite-directed mutagenesis and PCR.

The expression vectors containing nucleotide sequences encoding avariety of PKS enzymes for the production of different polyketides arethen transformed into the appropriate host cells to construct thelibrary. In one straightforward approach, a mixture of such vectors istransformed into the selected host cells and the resulting cells platedinto individual colonies and selected to identify successfultransformants. Each individual colony has the ability to produce aparticular PKS synthase and ultimately a particular polyketide.Typically, there will be duplications in some, most, or all of thecolonies; the subset of the transformed colonies that contains adifferent PKS in each member colony can be considered the library.Alternatively, the expression vectors can be used individually totransform hosts, which transformed hosts are then assembled into alibrary. A variety of strategies are available to obtain a multiplicityof colonies each containing a PKS gene cluster derived from thenaturally occurring host gene cluster so that each colony in the libraryproduces a different PKS and ultimately a different polyketide. Thenumber of different polyketides that are produced by the library istypically at least four, more typically at least ten, and preferably atleast 20, and more preferably at least 50, reflecting similar numbers ofdifferent altered PKS gene clusters and PKS gene products. The number ofmembers in the library is arbitrarily chosen; however, the degrees offreedom outlined above with respect to the variation of starter,extender units, stereochemistry, oxidation state, and chain lengthenables the production of quite large libraries.

Methods for introducing the recombinant vectors of the invention intosuitable hosts are known to those of skill in the art and typicallyinclude the use of CaCl₂ or agents such as other divalent cations,lipofection, DMSO, protoplast transformation, infection, transfection,and electroporation. The polyketide producing colonies can be identifiedand isolated using known techniques and the produced polyketides furthercharacterized. The polyketides produced by these colonies can be usedcollectively in a panel to represent a library or may be assessedindividually for activity.

The libraries of the invention can thus be considered at four levels:(1) a multiplicity of colonies each with a different PKS encodingsequence; (2) the proteins produced from the coding sequences; (3) thepolyketides produced from the proteins assembled into a function PKS;and (4) antibiotics or compounds with other desired activities derivedfrom the polyketides.

Colonies in the library are induced to produce the relevant synthasesand thus to produce the relevant polyketides to obtain a library ofpolyketides. The polyketides secreted into the media can be screened forbinding to desired targets, such as receptors, signaling proteins, andthe like. The supernatants per se can be used for screening, or partialor complete purification of the polyketides can first be effected.Typically, such screening methods involve detecting the binding of eachmember of the library to receptor or other target ligand. Binding can bedetected either directly or through a competition assay. Means to screensuch libraries for binding are well known in the art. Alternatively,individual polyketide members of the library can be tested against adesired target. In this event, screens wherein the biological responseof the target is measured can more readily be included. Antibioticactivity can be verified using typical screening assays such as thoseset forth in Lehrer et al., 1991, J. Immunol. Meth. 137: 167-173,incorporated herein by reference, and in the Examples below.

The invention provides methods for the preparation of a large number ofpolyketides. These polyketides are useful intermediates in formation ofcompounds with antibiotic or other activity through hydroxylation,epoxidation, and glycosylation reactions as described above. In general,the polyketide products of the PKS must be further modified, typicallyby hydroxylation and glycosylation, to exhibit antibiotic activity.Hydroxylation results in the novel polyketides of the invention thatcontain hydroxyl groups at C-6, which can be accomplished using thehydroxylase encoded by the eryF gene, and/or C-12, which can beaccomplished using the hydroxylase encoded by the picK or eryK gene.Also, the oleP gene is available in recombinant form, which can be usedto express the oleP gene product in any host cell. A host cell, such asa Streptomyces host cell or a Saccharopolyspora erythraea host cell,modified to express the oleP gene thus can be used to producepolyketides comprising the C-8-C-8a epoxide present in oleandomycin.Thus the invention provides such modified polyketides. The presence ofhydroxyl groups at these positions can enhance the antibiotic activityof the resulting compound relative to its unhydroxylated counterpart.

Methods for glycosylating the polyketides are generally known in theart; the glycosylation may be effected intracellularly by providing theappropriate glycosylation enzymes or may be effected in vitro usingchemical synthetic means as described herein and in PCT publication No.WO 98/49315, incorporated herein by reference. Preferably, glycosylationwith desosamine, mycarose, and/or megosamine is effected in accordancewith the methods of the invention in recombinant host cells provided bythe invention. In general, the approaches to effecting glycosylationmirror those described above with respect to hydroxylation. The purifiedenzymes, isolated from native sources or recombinantly produced may beused in vitro. Alternatively and as noted, glycosylation may be effectedintracellularly using endogenous or recombinantly produced intracellularglycosylases. In addition, synthetic chemical methods may be employed.

The antibiotic modular polyketides may contain any of a number ofdifferent sugars, although D-desosamine, or a close analog thereof, ismost common. Erythromycin, picromycin, megalomicin, narbomycin, andmethymycin contain desosamine. Erythromycin also contains L-cladinose(3-O-methyl mycarose). Tylosin contains mycaminose (4-hydroxydesosamine), mycarose and 6-deoxy-D-allose. 2-acetyl-1-bromodesosaminehas been used as a donor to glycosylate polyketides by Masamune et al.,1975, J. Am. Chem. Soc. 97: 3512-3513. Other, apparently more stabledonors include glycosyl fluorides, thioglycosides, andtrichloroacetimidates; see Woodward et al., 1981, J. Am. Chem. Soc. 103:3215; Martin et al., 1997, J. Am. Chem. Soc. 119: 3193; Toshima et al.,1995, J. Am. Chem. Soc. 117: 3717; Matsumoto et al., 1988, TetrahedronLett. 29: 3575. Glycosylation can also be effected using the polyketideaglycones as starting materials and using Saccharopolyspora eythraea orStreptomyces venezuelae or other host cell to make the conversion,preferably using mutants unable to synthesize macrolides, as discussedabove.

Thus, a wide variety of polyketides can be produced by the hybrid PKSenzymes of the invention. These polyketides are useful as antibioticsand as intermediates in the synthesis of other useful compounds. In oneimportant aspect, the invention provides methods for making antibioticcompounds related in structure to erythromycin, a potent antibioticcompound. The invention also provides novel ketolide compounds,polyketide compounds with potent antibiotic activity of significantinterest due to activity against antibiotic resistant strains ofbacteria. See Griesgraber et al., 1996, J. Antibiot. 49: 465-477,incorporated herein by reference. Most if not all of the ketolidesprepared to date are synthesized using erythromycin A, a derivative of6-dEB, as an intermediate. See Griesgraber et al., supra; Agouridas etal., 1998, J. Med. Chem. 41: 4080-4100, U.S. Pat. Nos. 5,770,579;5,760,233; 5,750,510; 5,747,467; 5,747,466; 5,656,607; 5,635,485;5,614,614; 5,556,118; 5,543,400; 5,527,780; 5,444,051; 5,439,890;5,439,889; and PCT publication Nos. WO 98/09978 and 98/28316, each ofwhich is incorporated herein by reference.

As noted above, the hybrid PKS genes of the invention can be expressedin a host cell that contains the desosamine, megosamine, and/or mycarosebiosynthetic genes and corresponding transferase genes as well as therequired hydroxylase gene(s), which may be either picK, megK, or eryK(for the C-12 position) and/or megF or eryF (for the C-6 position). Theresulting compounds have antibiotic activity but can be furthermodified, as described in the patent publications referenced above, toyield a desired compound with improved or otherwise desired properties.Alternatively, the aglycone compounds can be produced in the recombinanthost cell, and the desired glycosylation and hydroxylation steps carriedout in vitro or in vivo, in the latter case by supplying the convertingcell with the aglycone, as described above.

As described above, there are a wide variety of diverse organisms thatcan modify compounds such as those described herein to provide compoundswith or that can be readily modified to have useful activities. Forexample, Saccharopolyspora erythraea can convert 6-dEB to a variety ofuseful compounds. The compounds provided by the present invention can beprovided to cultures of Saccharopolyspora erythraea and converted to thecorresponding derivatives of erythromycins A, B, C, and D in accordancewith the procedure provided in the Examples, below. To ensure that onlythe desired compound is produced, one can use an S. erythraea eryAmutant that is unable to produce 6-dEB but can still carry out thedesired conversions (Weber et al., 1985, J. Bacteriol. 164(1): 425-433).Also, one can employ other mutant strains, such as eryB, eryC, eryG,and/or eryK mutants, or mutant strains having mutations in multiplegenes, to accumulate a preferred compound. The conversion can also becarried out in large fermentors for commercial production. Each of theerythromycins A, B, C, and D has antibiotic activity, althougherythromycin A has the highest antibiotic activity. Moreover, each ofthese compounds can form, under treatment with mild acid, a C-6 to C-9hemiketal with motilide activity. For formation of hemiketals withmotilide activity, erythromycins B, C, and D, are preferred, as thepresence of a C-12 hydroxyl allows the formation of an inactive compoundthat has a hemiketal formed between C-9 and C-12.

Thus, the present invention provides the compounds produced byhydroxylation and glycosylation of the compounds of the invention byaction of the enzymes endogenous to Saccharopolyspora erythraea andmutant strains of S. erythraea. Such compounds are useful as antibioticsor as motilides directly or after chemical modification. For use asantibiotics, the compounds of the invention can be used directly withoutfurther chemical modification. Erythromycins A, B, C, and D all haveantibiotic activity, and the corresponding compounds of the inventionthat result from the compounds being modified by Saccharopolysporaerythraea also have antibiotic activity. These compounds can bechemically modified, however, to provide other compounds of theinvention with potent antibiotic activity. For example, alkylation oferythromycin at the C-6 hydroxyl can be used to produce potentantibiotics (clarithromycin is C-6-O-methyl), and other usefulmodifications are described in, for example, Griesgraber et al., 1996,J. Antibiot. 49: 465-477, Agouridas et al., 1998, J. Med. Chem. 41:4080-4100, U.S. Pat. Nos. 5,770,579; 5,760,233; 5,750,510; 5,747,467;5,747,466; 5,656,607; 5,635,485; 5,614,614; 5,556,118; 5,543,400;5,527,780; 5,444,051; 5,439,890; and 5,439,889; and PCT publication Nos.WO 98/09978 and 98/28316, each of which is incorporated herein byreference.

For use as motilides, the compounds of the invention can be useddirectly without further chemical modification. Erythromycin and certainerythromycin analogs are potent agonists of the motilin receptor thatcan be used clinically as prokinetic agents to induce phase III ofmigrating motor complexes, to increase esophageal peristalsis and LESpressure in patients with GERD, to accelerate gastric emptying inpatients with gastric paresis, and to stimulate gall bladdercontractions in patients after gallstone removal and in diabetics withautonomic neuropathy. See Omura et al., 1987, Macrolides withgastrointestinal motor stimulating activity, J. Med. Chem. 30: 1941-3).The corresponding compounds of the invention that result from thecompounds of the invention being modified by Saccharopolyspora erythraeaalso have motilide activity, particularly after conversion, which canalso occur in vivo, to the C-6 to C-9 hemiketal by treatment with mildacid. Compounds lacking the C-12 hydroxyl are especially preferred foruse as motilin agonists. These compounds can also be further chemicallymodified, however, to provide other compounds of the invention withpotent motilide activity.

Moreover, and also as noted above, there are other useful organisms thatcan be employed to hydroxylate and/or glycosylate the compounds of theinvention. As described above, the organisms can be mutants unable toproduce the polyketide normally produced in that organism, thefermentation can be carried out on plates or in large fermentors, andthe compounds produced can be chemically altered after fermentation. Inaddition to Saccharopolyspora erythraea, Streptomyces venezuelae, S.narbonensis, S. antibioticus, Micromonospora megalomicea, S. fradiae,and S. thermotolerans can also be used. In addition to antibioticactivity, compounds of the invention produced by treatment with M.megalomicea enzymes can have antiparasitic activity as well. Thus, thepresent invention provides the compounds produced by hydroxylation andglycosylation by action of the enzymes endogenous to S. erythraea, S.venezuelae, S. narbonensis, S. antibioticus, M. megalomicea, S. fradiae,and S. thermotolerans.

The compounds of the invention can be isolated from the fermentationbroths of these cultured cells and purified by standard procedures. Thecompounds can be readily formulated to provide the pharmaceuticalcompositions of the invention. The pharmaceutical compositions of theinvention can be used in the form of a pharmaceutical preparation, forexample, in solid, semisolid, or liquid form. This preparation willcontain one or more of the compounds of the invention as an activeingredient in admixture with an organic or inorganic carrier orexcipient suitable for external, enteral, or parenteral application. Theactive ingredient may be compounded, for example, with the usualnon-toxic, pharmaceutically acceptable carriers for tablets, pellets,capsules, suppositories, solutions, emulsions, suspensions, and anyother form suitable for use.

The carriers which can be used include water, glucose, lactose, gumacacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc,corn starch, keratin, colloidal silica, potato starch, urea, and othercarriers suitable for use in manufacturing preparations, in solid,semi-solid, or liquified form. In addition, auxiliary stabilizing,thickening, and coloring agents and perfumes may be used. For example,the compounds of the invention may be utilized with hydroxypropylmethylcellulose essentially as described in U.S. Pat. No. 4,916,138,incorporated herein by reference, or with a surfactant essentially asdescribed in EPO patent publication No. 428,169, incorporated herein byreference.

Oral dosage forms may be prepared essentially as described by Hondo etal., 1987, Transplantation Proceedings XIX, Supp. 6: 17-22, incorporatedherein by reference. Dosage forms for external application may beprepared essentially as described in EPO patent publication No. 423,714,incorporated herein by reference. The active compound is included in thepharmaceutical composition in an amount sufficient to produce thedesired effect upon the disease process or condition.

For the treatment of conditions and diseases caused by infection, acompound of the invention may be administered orally, topically,parenterally, by inhalation spray, or rectally in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvant, and vehicles. The term parenteral, asused herein, includes subcutaneous injections, and intravenous,intramuscular, and intrasternal injection or infusion techniques.

Dosage levels of the compounds of the invention are of the order fromabout 0.01 mg to about 50 mg per kilogram of body weight per day,preferably from about 0.1 mg to about 10 mg per kilogram of body weightper day. The dosage levels are useful in the treatment of theabove-indicated conditions (from about 0.7 mg to about 3.5 mg perpatient per day, assuming a 70 kg patient). In addition, the compoundsof the invention may be administered on an intermittent basis, i.e., atsemi-weekly, weekly, semi-monthly, or monthly intervals.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, aformulation intended for oral administration to humans may contain from0.5 mg to 5 gm of active agent compounded with an appropriate andconvenient amount of carrier material, which may vary from about 5percent to about 95 percent of the total composition. Dosage unit formswill generally contain from about 0.5 mg to about 500 mg of activeingredient. For external administration, the compounds of the inventionmay be formulated within the range of, for example, 0.00001% to 60% byweight, preferably from 0.001% to 10% by weight, and most preferablyfrom about 0.005% to 0.8% by weight.

It will be understood, however, that the specific dose level for anyparticular patient will depend on a variety of factors. These factorsinclude the activity of the specific compound employed; the age, bodyweight, general health, sex, and diet of the subject; the time and routeof administration and the rate of excretion of the drug; whether a drugcombination is employed in the treatment; and the severity of theparticular disease or condition for which therapy is sought.

A detailed description of the invention having been provided above, thefollowing examples are given for the purpose of illustrating theinvention and shall not be construed as being a limitation on the scopeof the invention or claims.

EXAMPLE 1 Production of Methylmalonyl-CoA in E. Coli

This example describes, in part A, the cloning and expression ofmethylmalonyl-CoA mutase, and in part B, the cloning and expression ofmethylmalonyl-CoA epimerase, in E. coli.

A. Cloning and Expression of Methylmalonyl-CoA Mutase

Methylmalonyl-CoA mutase was cloned from Propionibacterium shermanii andexpressed in E. coli. The holoenzyme mm-CoA mutase was obtained bygrowing cells in the presence of hydroxocobalamin and was shown to beactive without addition of vitamin B12. Methylmalonyl-CoA was producedin vivo, as seen by CoA analysis using a panD strain of BL21 (DE3).

To support modular polyketide production in E. coli, the inventionprovides methods and reagents to produce (S)-methylmalonyl-CoA, which isnot naturally present in E. coli, by overexpressing mm-CoA mutase andmm-CoA epimerase in E. coli. An active, FLAG-tagged version of themm-CoA mutase from S. cinnamonensis was expressed in XL1Blue cells,which were grown in the presence of hydroxocobalamin in a synthetic,vitamin-free media to produce active holoenzyme. The CoA levels in thecells were analyzed by feeding labeled β-alanine; for this purpose it isbeneficial to have a panD strain, which is a β-alanine auxotroph. Themutase DNA rearranged in the panD strain of SJ16, a recA⁺ strain, suchthat the CoA analysis had to be carried out without the panD. Thisresulted in a lower signal to noise ratio, but elevated mm-CoA levelscould still be detected. As an alternative to the S. cinnamonensisgenes, the invention provides a mm-CoA mutase from P. shermanii clonedinto an E. coli expression vector, which is active without addition ofvitamin B12, and which elevates mm-CoA levels in E. coli in a panDstrain compatible with the mutase DNA.

Propionibacterium freudenreichii subsp. shermanii was obtained as a stabin tomato juice agar from derived from a freeze-dried specimen fromNCIMB, Scotland (NCIMB # 9885). E. coli strain gg3, a panD version ofBL21 (DE3) was used for the CoA analysis. E. coli strains gg1 and gg2,recA⁻ versions of the SJ16 panD strain, were also used. The vector pKK**is a version of pKK223-3 in which the cloning region is altered to rangefrom Nde1 to EcoR1 and an extra Nde1 site is deleted. Growth of P.shermanii and preparation of genomic DNA was conducted as described inthe literature.

Subcloning of methylmalonyl-CoA mutase from P. shermanii into E. coliwas conducted as follows. The gene for mm-CoA mutase consists of twosubunits, mutA and mutB, which were amplified by PCR from P. shermaniigenomic DNA in a total of four fragments. Naturally occurringrestriction sites were used to piece the gene together. Uniquerestriction sites were introduced at both ends of the gene for cloningpurposes, and the start codon for the mutB gene was changed from GTG toATG. As illustrated below, these four fragments were cloned into aBluescript™ (Stratagene) vector, sequenced, and then pieced together toform the complete mutase gene. The gene was then cloned into expressionvectors pET22b and pKK** between the restriction sites Nde1 and HindIII,to form pET-MUT and pKK**-MUT.

The pET-MUT was transformed into competent cells BL21(DE3) and laterinto cells gg3, which are apanD version of BL21(DE3). The pKK**-MUT wastransformed into SJ16 panD and into XL1Blue. The DNA was tested byscreening several colonies with Nde1 and HindIII, to determine if themutase gene was still present or if it had rearranged.

For SDS-PAGE analysis, cells of strain BL21(DE3) containing pET-MUT (andpET alone, as a control) were grown aerobically at 27° C. in MUT mediawith 100 μg/ml carbenicillin (carb) (MUT media is M9 salts, glucose,thiamine, trace elements and amino acids, as previously described forthe expression of methionine synthase (Amaratunga, M., et al., Asynthetic module for the metH gene permits facile mutagenesis of thecobalam in-binding region of Escherichia coli methionine synthase:initial characterization of seven mutant proteins. Biochemistry, 1996.35(7): p. 2453-63). Overnight cultures (250 μl) were used to inoculate25 mL of MUT media (carb), which were grown at 27° C. to an OD₆₀₀ ofapproximately 0.5. The cultures were then induced with IPTG to 1 mMfinal concentration. Two cultures were left at 27° C. for three hourswhile duplicate cultures were grown at 37° C. for two hours. The cellswere collected by centrifugation and the pellets were stored at −80° C.prior to analysis. The cells were lysed by sonication and both thesoluble and insoluble phases were examined by SDS/PAGE. This procedurewas repeated for cells of strain XL1Blue containing pKK**-MUT.

For expression of active mm-CoA mutase (with hydroxocobalamin), cells ofstrain gg3 containing pET-MUT (and pET alone, as a control) were grownin MUT media (carb) and 5 μM beta-alanine for approximately 20 hours at27° C. The following operations were performed in a dark room with a redsafelight: 125-mL flasks, each containing 25 mL of MUT media with carband 5 μM β-alanine and wrapped in aluminum foil, were inoculated with 5μM hydroxocobalamin and then with 250 μL from the respective startercultures. After shaking overnight at 27° C., the cultures were inducedwith IPTG to 1 mM final concentration and grown for an additional 4:45hours, at which point they were collected (in Falcon tubes wrapped inaluminum foil) by centrifugation at 4000 rpm for 10 minutes. The pelletswere stored in the dark at −80° C. prior to assaying.

The mutase assay was performed as follows. All operations were performedin the dark or under a red safelight. The pellet from 25 mL of culturewas thawed, washed in buffer C (50 mM potassium phosphate pH 7.4, 5 mMEDTA, 10% glycerol), and resuspended in 0.5 mL of buffer C containingprotease inhibitors (1 tablet per 10 mL of buffer). Following sonicationon ice, the extract was clarified by centrifugation at 4° C. for 10minutes at maximum speed in an Eppendorf microfuge; the supernatent wasassayed. Enzyme assays contained, in a final volume of 100 μL, 0.2 mM(2R,2S)-methylmalonyl-CoA, mutase extract, and buffer C containingprotease inhibitors. Reactions for assays with vitamin B12 were as abovebut contained 0.01 mM vitamin B12, in which case the mutase extract wasincubated with the vitamin B12 in a total volume of 75 μL for 5 minutesat 30° C. prior to initiation of reaction with methylmalonyl-CoA. Afterthe desired length of incubation at 30° C., the reaction was stopped bythe addition of 50 μL of 10% trichloroacetic acid (TCA) and placed onice for approximately 10 minutes. Cellular debris and precipitatedprotein were removed by centrifugation for 5 minutes in an Eppendorfmicrofuge at 4° C. An aliquot (100 μL) of the supernatant was injectedonto the HPLC to quantify conversion of methylmalonyl-CoA tosuccinyl-CoA. One time point was taken after 20 minutes of incubation at30° C., and the sample was assayed for conversion of mm-CoA tosuccinyl-CoA. All operations were performed exclusively under a redsafelight until the reaction was stopped by addition of TCA.

The CoA analysis was performed as described in the literature, exceptthat 5 μM of hydroxocobalamin were added at the time of IPTG induction,and the tubes were wrapped in aluminum foil and grown at 27° C. insteadof 30° C. The CoA peaks, which eluted in approximately one minute each,were collected manually, as well as approximately one minute of sampleboth before and after each peak. In some tests, fractions were collectedevery 30 seconds. All samples were counted in the scintillation counter.

The two subunits of the gene encoding methylmalonyl-CoA mutase aretranslationally coupled—the GTG start codon of the downstream subunitmutB overlaps with the ATG codon of mutA. The GTG valine start wasmutated to an ATG methionine start (which does not alter any other aminoacids), because E. coli utilizes the methionine start more efficiently.Sequencing the mm-CoA mutase gene revealed a discrepancy between thesequence observed and the published sequence. A “GC” instead of a “CG”changed two amino acids from Asp, Val to Glu, Leu. The crystal structureof mm-CoA mutase from P. shermanii showed that the two amino acids areindeed Glu, Leu, so the published sequence is in error. The mm-CoAmutase gene was subcloned into two different E. coli expression systems:pET, which is under control of the strong T7 promoter, and pKK, whichuses the leaky tac promoter. First it was necessary to find strains inwhich the mutase DNA did not rearrange. It was previously observed thata FLAG-tagged version of the mutase from S. cinnamonensis rearranged inSJ16 panD and in BL21(DE3), which are both recA⁺ strains, but not inXL1Blue, which is recA⁻. This mutase DNA (P. shermanii) also rearrangedin the SJ16 cells but not in the BL21(DE3) cells. Thus a panD version ofBL21(DE3) was created (gg3) for use with the pET vector. A recA⁻ versionof SJ16 was also created (gg1, gg2) for use with the pKK system;however, the mutase DNA rearranged in this strain as well.

Different growth conditions were tested to find conditions in which thetwo subunits of the mutase were expressed in the soluble phase inapproximately equal molar ratios. In general, it seemed that the highertemperature of 37° C. caused the mutase to appear predominantly in theinsoluble form. Growth exclusively at 27° C. resulted in soluble proteinwith an approximately equal subunit ratio.

FIG. 3 shows the comparison of in vivo acyl-CoA levels in BL21(DE3)panDstrains with and without mm-CoA mutase. For each CoA, the ratio ofthe amount in the strain containing the mutase to the amount in thecontrol strain was determined. Interestingly, malonyl-CoA was increasedabout 25-fold and succinyl-CoA about 3-fold. Acetyl-CoA and CoA wereincreased just slightly, and propionyl-CoA was not detected in eithercase.

To express active mutase in vivo, it was necessary to grow cells in adefined media (MUT media) that allows uptake of the vitamin B12precursor hydroxocobalamin; this is similar to an established protocolfor expression of active methionine synthase, which also requires B12.Cell extracts overexpressing the mutase were shown to convert mm-CoA tosuccinyl CoA without the addition of vitamin B12. Only one time point(at 20 minutes) was assayed to confirm activity; the specific activityof the mutase was not determined.

Thus, methylmalonyl-CoA mutase was expressed as the active holoenzyme inE. coli, and methylmalonyl-CoA was produced in vivo. Because a slow,spontaneous chemical epimerization between (R)- and (S)-mm-CoA doesexist (approximately 3% in 15 minutes), it may be helpful to determinethe relative amounts of these diastereomers in cells overexpressing themutase. Enough (S)-mm-CoA may be present to support polyketideproduction in some cells without addition of an epimerase. To facilitatethe eventual production of polyketides in E. coli, the mutase gene canbe incorporated into the chromosome of the BL21 panD cell or other hostcell.

FIG. 2 shows the construction of pSK-MUT, in which four PCR fragmentswere sequenced and pieced together to form the complete mutase gene inpSK-bluescript.

In follow-up experiments, the specific activity of the mutase wasdetermined and an in-depth CoA analysis was completed. As shown in FIG.3, the CoA levels in the cells were again analyzed using a panD strain,which is a β-alanine auxotroph. ³H-β-alanine was fed to the cells andincorporated into the acyl-CoAs, which were separated via HPLC andcounted. The CoA pools for cell extracts with and without the mutase, aswell as with and without hydroxocobalamin, were examined.

To test whether acyl-CoAs degrade in TCA, the following tests wereconducted. The CoA mix consisted of 1.6 mM each of malonyl-,methylmalonyl-, succinyl-, acetyl-, and propionyl-CoA, plus 0.5 mM CoA.An aliquot (10 μL) of this mix was added to 100 μL 10% TCA, 50 μL wereimmediately injected to the HPLC for CoA analysis, and the remainder waspromptly frozen on dry ice. The frozen portion was then thawed andloaded immediately to the HPLC. Again, 10 μL of the CoA mix were addedto 100 μL 10% TCA, 50 μL were left on ice for 15 minutes and theninjected to the HPLC, the remainder was left at 4° C. overnight andinjected to the HPLC the next morning. The area under each CoA peak wasnoted. This same procedure was followed but using a mixture of TCA andbuffer A from the mutase assay.

The CoA analysis described here is carried out on cells which are lysedin 10% TCA. Thus, determining whether the CoAs degrade significantly inTCA and in a mixture of TCA and buffer A from the mutase assay isimportant. The tests showed that the percent of each CoA relative to thetotal CoA pool, as well as the overall amount of CoA, remained constantafter freeze/thawing, after leaving on ice for 15 minutes, and afterleaving the sample overnight at 4° C. Thus, the CoAs are stable in TCAand in the mutase assay buffer after the cells are lysed or after theassays are completed, and prior to HPLC analysis.

Although the CoAs are stable in TCA and buffer at 4° C., they degradedat 30° C., the temperature at which the mutase assay was performed. Infive minutes under the assay conditions, about 4% of themethylmalonyl-CoA hydrolyzed to CoA. The succinyl-CoA hydrolyzed at acomparable rate. Thus, the mutase assay is suboptimal for extremelyquantitative results.

When 0.2 mM methylmalonyl-CoA was incubated with a crude lysate fromcell extracts overexpressing the mutase, succinyl-CoA was produced. Nosuccinyl-CoA was observed when methylmalonyl-CoA was incubated withlysates from the control strain (containing the plasmid vector butlacking the mutase genes). Under these expression and assay conditions,a specific activity of approximately 0.04 U/mg was observed in the crudeextracts. When cells overexpressing the mutase were grown in MUT mediawithout hydroxocobalamin, no mutase activity was observed; however,mutase activity could be detected by addition of vitamin B12 in vitro.Adding vitamin B12 to extracts that were grown in the presence ofhydroxocobalamin resulted in increased mutase activity, suggesting thata significant amount of expressed mutase is present as the apo-enzyme.This might have occurred because the enzyme was expressed faster thanthe hydroxocobalamin could be transported into the cell, or because thevitamin B12 cofactor was lost during preparation of the extract.

FIG. 4 shows the comparison of in vivo acyl-CoA levels with and withoutthe mutase and with and without hydroxocobalamin. In the cellsoverexpressing the mutase and grown with hydroxocobalamin,methylmalonyl-CoA comprised 13% of the overall CoA pool, whereas in theother cells no methylmalonyl-CoA was detectable. The background level ofcounts is about 0.25% of the overall number of counts in the CoAs,suggesting that any methymalonyl-CoA present in E. coli strains notoverexpressing the mutase would comprise at most 0.25% of the overallCoA pool, or 2% of the amount of methylmalonyl-CoA observed in thestrain overexpressing the mutase. The composition of the CoA poolobserved for the E. coli panD strain is consistent with that observedpreviously for E. coli panD mutants grown on glucose.

Thus, the methylmalonyl-CoA mutase from P. shermanii has beenoverexpressed as the active holoenzyme in E. coli and shown to produce(2R)-methylmalonyl-CoA in vivo. Conversion of (2R)- to(2S)-methylmalonyl-CoA via methylmalonyl-CoA epimerase should provide anadequate supply of the correct isomer of methylmalonyl-CoA to supportheterologous production of complex polyketides E. coli.

FIG. 4 shows the results of CoA analysis of E. coli overexpressingmethylmalonyl-CoA mutase. The levels of ³H detected in fractionscollected from HPLC of cell-free extracts from ³H β-alanine-fed E. coliharboring either the pET control vector grown without hydroxocobalamin(solid line), pET grown with hydroxocobalamin (dash-dotted), pEToverexpressing the mutase and grown without hydroxocobalamin (dotted),or pET overexpressing the mutase and grown with hydroxocobalamin(dashed) are shown.

B. Cloning and Expression of Methylmalonyl-CoA Epimerase

The mm-CoA epimerase from Propionibacterium shermanii was purified andused to obtain N-terminal protein sequence as well as internal peptidesequence from LysC-generated peptides. The epimerase gene was clonedusing hybridization probes designed from the peptide sequences.

Propionibacterium freudenreichii subsp. shermanii was obtained andcultured as described in part A. Purification of mm-CoA epimerase fromP. shermanii was based on a modification of the published procedure. Theprocedure utilized a 10 L culture, which was lysed by sonicationfollowed by column chromatography in the order: DE-52, Hydroxyapatite,Phenylsepharose, MonoQ anion exchange, and C-8 RP HPLC.

All operations were performed at 4° C., except the C-8 RP HPLC, whichwas performed at room temperature, and all buffers contained 0.1 mMPMSF, unless otherwise stated. The epimerase assay was performedessentially as described in the literature. Protein concentration wasdetermined using the method of Bradford. The overall yield of epimeraseactivity was not determined.

More specifically, cell paste (75 g) was resuspended in 50 mL buffer (50mM Tris-HC1 pH 7.5, 0.1M KCl, 0.2 mM PMSF, 1 mM EDTA) and sonicatedusing a macrotip with a diameter of 1.2 cm. With pulses of 0.5 secondsON and 0.3 seconds OFF, the cells were sonicated twice for 30 secondseach at a power setting of 4, followed by five times for 30 seconds eachat a power setting of 6. A clear, amber-colored supernatant (53.5 ml)was obtained after spinning for 35 minutes at 12,000 rpm.

The crude extract from above was applied to a column (diameter 2.5 cm,height 15 cm) of 73 mL of DE-52 resin equilibrated with 50 mM Tris-HClpH 7.5, 0.1M KCl. The column was washed at 1 ml/min with three columnvolumes of the above buffer, followed by a linear gradient to 50 mMTris-HCl pH 7.5, 0.5 M KCl over seven column volumes. Six mL fractionswere collected and assayed for epimerase activity. The epimerase wasfound predominantly in the flow-through and in several early fractions.The flow-through and active fractions were combined (325 mL) anddialyzed against 4 liters of 50 mM Tris-HCl pH 7.5, 10% glycerol,followed by 4 liters of 10 mM sodium phosphate pH 6.5, 10% glycerol(final volume 250 mL).

A 7.5 mL hydroxyapatite biogel HTP gel column (diameter 1.5 cm, height16 cm) was equilibrated with 10 mM sodium phosphate pH 6.5, 5% glycerol.After loading of the enzyme solution (using repeated injections) andwashing with three column volumes of the above buffer, a gradient to 200mM sodium phosphate pH 6.5, 5% glycerol was effected over 20 columnvolumes at a flow rate of 1 ml/min. The 2 mL fractions were assayed forepimerase activity, and fractions containing epimerase activity werepooled for a total of 99 ml.

To the 99 mL sample from above, solid ammonium sulfate to 1.5 M finalconcentration was added slowly and with stirring at 4° C. over 30minutes. This suspension (100 mL) was loaded, by repeated injection,onto a 6.6 mL column (1 cm×height 8.5 cm) of phenyl-sepharose resinequilibrated in 20 mM sodium phosphate buffer pH 6.5, 1.5 M ammoniumsulfate. The column was washed at 1 ml/min with three column volumes ofthis buffer, followed by a linear gradient to 20 mM sodium phosphatebuffer pH 6.5, 10% glycerol, over 24 column volumes. After assaying the3 mL fractions for epimerase activity, the fractions containingepimerase activity were pooled and dialyzed against 50 mM Tris-HCl pH7.5.

A mono Q 5/5 prepacked column was equilibrated with 25 mM Tris-HCl pH7.5 at 0.5 mL/min. The sample from the previous step was loaded onto thecolumn, which was then washed with 5 column volumes of the above buffer,followed by a linear gradient to 50 mM Tris-HCl pH 7.5, 1 M NaCl, 5%glycerol, over 50 column volumes. The 1 mL fractions were assayed forepimerase activity. Several fractions containing epimerase activity werestored separately; the fraction with the most activity was used for thenext purification step.

A reverse-phase column was equilibrated with water containing 0.1%trifluoroacetic acid; 120 μL (concentrated from 0.5 mL of the activefraction from above, using an Amicon microconcentrator) was injectedonto the column at a flow rate of 0.2 mL/min and washed for five minuteswith the above solvent system. Then a linear gradient over 50 minutes toacetonitrile containing 0.1% trifluoroacetic acid was implemented. Thepeaks were collected manually and the peak corresponding to theepimerase (as determined by SDS/PAGE) was dried to completeness,resuspended in water and stored at −80° C.

For Lys C mediated digestion of the HPLC-purified epimerase, theepimerase fraction (11751rp2-B, 200 μL) collected from reverse phaseHPLC was dried to completeness and resuspended in 40 μL water. To 30 μLof the sample was added 5 μL of 1 M Tris/HCl, pH 8, 1.5 μL of 0.1 M DTT,2 μL of Lys C protease (0.2 μg). A control reaction contained all of theabove components except the epimerase. The reactions were incubatedovernight at 37° C. An aliquot of the reaction (5 μL) was diluted to 60μL with water and loaded to the HPLC, using the same HPLC program thatwas used to purify the epimerase. The analytical HPLC showed that theLys C digestion was not complete. An additional aliquot of Lys C (0.2μg) was added to the reactions and incubation was continued overnight at37° C. Following overnight incubation, an aliquot of the reaction (5 μL)was diluted to 60 μL with water and subjected to the HPLC. The HPLCshowed that the digestion was complete. The remainder of the reactionwas loaded to the HPLC and individual peaks were collected manually.HPLC of the control reaction showed no significant peptide fragmentsarising from self-digestion of the LysC.

An aliquot of the pure epimerase, as well as a peptide collected fromthe procedure described above, were submitted for N-terminal amino acidsequencing. Based on the amino acid sequences from above, severaldegenerate primers were designed as described below that introducedunique restriction sites to either end of the eventual PCR product.These primers were used in PCR with P. shermanii genomic DNA to obtain a200 base-pair product, which was cloned into a Bluescript™ (Stratagene)vector and submitted for sequencing.

A cosmid library of P. shermanii was prepared, essentially as describedin the Stratagene cosmid manual. The titer of this cosmid library wasapproximately 11 cfu (colony forming units) per μL, for a total yield of5556 cfu. A plasmid library of P. shermanii was prepared by digesting P.shermanii genomic DNA with SacI and ligating the resulting mixture intoa Bluescript™ vector also cut with SacI. To determine the average insertsize (2 kb), ten random clones were digested with SacI. The ligationmixture was re-transformed 5 times, pooled and plated on one large LB(carb) plate, resulting in a lawn of colonies that were scraped togetherand resuspended in LB as the plasmid library. The titer of this plasmidlibrary was approximately 64,000 cfu per μL.

Several degenerate primers based on the amino acid sequences wereprepared and used in PCR with P. shermanii genomic DNA to obtain a 180base-pair product, which was cloned into a Bluescript™ vector andsequenced. Several different probes were made. The first probe was madeusing the random priming method to incorporate either ³²P or digoxigenininto the epimerase fragment. A probe was made from the cloned fragmentby amplification of the fragment via PCR, using the digoxigenin labelingmethod. The PCR product was gel isolated, quantified, and used to probethe cosmid library. Colonies that hybridized to the probe wererestreaked from master plates, and five colonies from the re-streakedplates were picked, cosmids were isolated, and the insert sequencesscreened for the epimerase gene by PCR. Several cosmids that were scoredpositive for epimerase DNA sequence by PCR were subjected to DNAsequencing using epimerase-specific primers. The cosmid designated117-167-A7 contained the full epimerase sequence.

The sequence of the putative epimerase gene contained in cosmid117-167-A7 was aligned to the N-terminal epimerase sequence alreadyknown. The several hundred base pairs downstream of this sequence weretranslated in all three frames and a stop codon in one of the frames wasfound that yielded a protein of the expected size. The entire sequencewas used to search the protein database via BLAST analysis, and thesequence showed high homology to the sequence of a putative epimerasefrom S. coelicolor identified in accordance with the methods of theinvention. PCR primers were designed based on the DNA sequence of thecloned P. shermanii epimerase and the gene was amplified from P.shermanii genomic DNA with NdeI and BamHI sites at the 5′-end, aninternal NdeI site was destroyed near the 5′ end, and NheI and AvrIIsites were introduced at the 3′-end. Following PCR, the 447 bp productwas cloned into a Bluescript vector (143-6-11) and sequenced. Also, fouradditional sequencing primers were designed to provide several-foldcoverage of the epimerase gene. The full epimerase gene sequenceprovided in isolated and recombinant form by the present invention isprovided below as SEQ ID NO: 1, and the protein sequence of theepimerase is provided below as SEQ ID NO: 2.

The epimerase gene was then cloned into a pET expression vector; theconstruct was named pET-epsherm.

For the cloning of epimerase genes from B. subtilis (described by Halleret al., supra) and S. coelicolor (from cosmid 8F4 in the S. coelicolor,genome sequencing project), primers were designed to PCR these genesfrom their respective genomic DNAs and to incorporate either a PacI orNdeI site at the 5′ end, and an NsiI site at the 3′ end. The PCRproducts were cloned into a Bluescript™ vector and sequenced.Mutation-free clones were obtained for the S. coelicolor epimerase, butthe B. subtilis epimerase contained two point mutations in all threeclones tested: C to T at base pair 37, and G to A at base pair 158. Whenthe PCR for this epimerase gene was repeated and the product cloned andsequenced, the same mutations were present, implying that the originalsequence was in error. The cloned epimerases from B. subtilis and S.coelicolor were cloned as NdeI/NsiI fragments into an intermediatevector 116-172a, a Bluescript™ pET plasmid containing the T-7 promoterand terminator sequences. The cloned epimerases from B. subtilis and S.coelicolor are pET-epsub and pET-epcoel, respectively. The epimerasegenes were also excised along with the T7 promoter as PacI/NsiIfragments, as shown schematically below.

-   -   ---PacI---T7 promoter------epimerase gene--------NsiI---        and cloned into the PacI/NsiI restricted vector 133-9b, to form        a single operon with the epimerase gene located downstream of        the two mutase genes. The epimerase gene from P. shermanii was        cloned as above except that it was cloned into 116-172a as an        NdeI/AvrII fragment, excised along with the T7 promoter as a        PacI/NheI fragment, and cloned into 133-9b between PacI and NheI        sites. The constructs are pET-mutAB-T7-epsherm,        pET-mutAB-T7-epsub, and pET-mutAB-T7-epcoel.

As an alternative to the mutase from P. shermanii, S. coelicolor, and B.subtilis, one can clone by PCR from E. coli genomic DNA the single genefor Sbm (sleeping beauty mutase). Genomic DNA of E. coli BL21(DE3)/PanDwas prepared using a kit purchased from Qiagen. The gene for Sbm(Sleeping beauty mutase, a methylmalonyl-CoA mutase) was amplified byPCR from E. coli BL21(DE3)/PanD genomic DNA. The PCR fragment was gelisolated, cloned into PCRscript and sequenced to yield the mutation-freeclone 143-11-54. Excised as an NdeI/SacI fragment, sbm was cloned intopET22b, thence as a NdeI/XhoI fragment into pET16b to introduce anN-terminal His-Tag (143-49-2). Sbm was also cloned between NdeI and SpeIinto 116-95B.43, a pET22b vector that allows subsequent cloning of theepimerase genes downstream of the sbm. That construct was named143-40-39.

Cells of strain BL21(DE3) containing pET-epsherm, pET-epcoel, pET-epsub,or a control pET vector were grown overnight at 37° C. in 2 mL LBcontaining 100 μg/ml carbenicillin. The starter culture (250 μL) wasused to inoculate 25 mL LB containing 100 μg/ml carbenicillin. Thecultures were grown at 37° C. to an OD of approximately 0.4, theninduced with IPTG to 1 mM final concentration and grown for anadditional 3 hours at 30° C. The cells were collected by centrifugationat 4000 rpm for 10 minutes, and the pellets were stored at −80° C. priorto assay. The epimerase from P. shermanii expressed well in E. coli; SDSgel analysis revealed an overexpressed protein at approximately 22 kDa.The S. coelicolor epimerase also expressed well, at a molecular weightof approximately 19 kDa, and the B. subtilis epimerase was expressed,but mostly in inclusion bodies (a faint band is present at approximately19 kDa), which can be overcome by use of alternate expression systems.

Epimerase activity was measured in crude extracts of E. coli harboringeither pET-epsherm, pET-epcoel, pET-epsub, or a control pET vector. Theepimerase assay couples transcarboxylase, which converts(S)-methylmalonyl-CoA into propionyl-CoA, to malate dehydrogenase, whichconverts NADH into NAD⁺, producing a decrease in absorbance at 340 nm.The assay is initiated with a racemic mixture of(R,S)-methylmalonyl-CoA; when the (S)-isomer is consumed as describedbelow, a steady background rate is observed at about one-tenth of theinitial rate. When an extract containing epimerase is added to theassay, the (R)-isomer is converted to (S)-, resulting in a furtherdecrease in absorbance. In crude E. coli extracts, however, asignificant background rate is observed, probably due to an endogenousNADH oxidase. Thus the epimerase must be expressed at a sufficientlyhigh level to conclude that it is active. The assay was conducted asfollows.

The pellet from approximately 20 mL of culture was thawed andresuspended in 2 mL 1× assay buffer containing a protease inhibitorcocktail tablet. The cells were disrupted by sonication (two sonicationcycles for 30 seconds each at a power setting of 2 [pulse ON 0.5sec/pulse OFF 0.5 sec]). After spinning for 10 minutes at 13,000 rpm inan Eppendorf centrifuge, the supernatents were saved for assay.Methylmalonyl-CoA epimerase activity was assayed using a modification ofthe method of Leadlay et al., Biochem. J. 197: 413-419, “Purificationand characterization of methylmalonyl CoA epimerase fromPropionibacterium shermanii” (1981). The assays were performed at 30° C.with a 1 cm path length plastic cuvette, in a final volume of 1.5 mL.The reaction mixtures contained 0.2 M potassium phosphate buffer pH 6.9,0.1 M ammonium sulfate, 5 mM sodium pyruvate, 0.08 mM(2R,2S)-methylmalonyl-CoA, 0.05 units of partially purifiedtranscarboxylase, 0.16 mM NADH, and 2.5 units malate dehydrogenase. Thereaction was initiated with (2R,2S)-methylmalonyl-CoA and the decreasein absorbance at 340 nm was monitored, reflecting the disappearance ofthe 2S isomer. When the decrease in absorbance at 340 nm reached thebasal level (usually around 10% of the initial transcarboxylase rate),an extract containing epimerase was added and a further decrease inabsorbance was observed. The chemicals and enzymes used in the epimeraseassay were purchased from Sigma, except for transcarboxylase, which wasobtained as a crude preparation from Case Western Reserve.

The crude extracts harboring both the P. shermanii and S. coelicolorepimerases had specific activities (approximately 30 units/mg) at least10 times higher than that of the control. However, no activity above thebackground level was observed in the extract harboring the B. subtilisepimerase, possibly because it was not expressed at a high enough level,or as noted above, was expressed as insoluble inclusion bodies. ThepET-mutAB-T7-epsherm construct was also expressed in E. coli. Theresulting crude extract contained epimerase activity that wassignificantly above the background level; thus, the epimerase isfunctional in this construct. The mutase did not interfere in theepimerase assay, because these cells were grown without addition ofhydroxocobalamin, the cofactor for mutase activity. These results showthat one can express both active mutase and active epimerase in an E.coli cell. These results also show that the methylmalonyl-CoA epimerasefrom P. shermanii was cloned, expressed in E. coli, and active, and thatthe putative epimerase from S. coelicolor is a methylmalonyl-CoAepimerase. These genes can be integrated into the chromosome of an E.coli PanD strain or other strain and used for the production ofpolyketides built in whole or in part from methylmalonyl CoA.

EXAMPLE 2 Production of Methylmalonyl CoA in Yeast

This example describes the construction of strains of Saccharomycescerevisiae optimized for polyketide overproduction. In particular, thisexample describes the construction of yeast host strains that (i)produce substrates and post-translational modification enzymes necessaryto express polyketides made by modular polyketide synthases; (ii) havenecessary nutritional deficiencies to allow positive selection of atleast three compatible plasmids; and/or (iii) are suitable to permitradioactive labeling of acyl-CoA pools and polyketide synthases anddemonstrates that such strains can express a modular PKS and produce acomplex polyketide at levels suitable for commercial development.References are cited in this example by a number corresponding to thenumbered list of references below, each of which is incorporated hereinby reference.

With appropriate strain modifications, S. cerevisiae is an ideal hostfor polyketide production. S. cerevisiae is capable of producing veryhigh levels of polyketides. Introduction of the gene for the iterativePKS, 6-MSAS, along with the gene for Sfp, a P-pant transferase from B.subtilis, led to the production of an impressive 2 g/L 6-MSA inshake-flasks without optimization Kealey, J. T., et al., Production of apolyketide natural product in nonpolyketide-producing prokaryotic andeukaryotic hosts. Proc Natl Acad Sci USA, 1998. 95(2): p. 505-9,incorporated herein by reference. The genetics of yeast is very wellunderstood. Genes can readily be inserted into the chromosome, and thecomplete genome sequence provides relevant knowledge regarding metabolicpathways and neutral insertion sites. In addition, several strong,controllable promoters are available. Proteins have less tendency toform inclusion bodies in yeast, compared to E. coli. Yeast has arelatively short doubling time in comparison to native polyketideproducing organisms. S. cerevisiae has a doubling time of 1 to 2 hrcompared to 4 to 24 hr for a typical polyketide producer, which hasobvious benefits in genetic development, process development, andlarge-scale production.

The fact that yeast grow as single cells provides an additional benefitover filamentous organisms (typical polyketide producers). Mycelialfermentations are viscous and frequently behave as non-Newtonian fluids.This fluid rheology provides a significant obstacle to the processscientist both in terms of uniform nutrient transport to the cells andin handling the fermentation broth. Employing yeast as a host, even athigh cell densities, avoids such impediments. Because of the extensivehistory of yeast in single cell protein production and the expression ofrecombinant proteins, scalable fermentation protocols for yeast havebeen developed. Yeast can be grown in fed-batch fermentations to veryhigh cell densities (>100 g/L biomass) as compared to typical polyketideproducers (10-20 g/L biomass). Thus, comparing organisms with the samespecific productivity (g polyketide/g biomass/day), yeast would providea higher volumetric productivity (g polyketide/L/day). Finally, S.cerevisiae is classified by the FDA as a “Generally Regarded As Safe”(GRAS) organism. GRAS classification will facilitate approval of drugsproduced in yeast as compared to other host cells.

S. cerevisiae also has disadvantages as a host for polyketidebiosynthesis, most of which are related to the fact that yeast did notevolve to produce polyketides. Yeast does not contain methylmalonyl-CoA,a necessary precursor for biosynthesis of many polyketides. Yeast doesnot have a suitable P-pant transferase capable of the necessary posttranslational modification of ACP domains of a PKS. Yeast codons arebiased towards A+T, whereas most polyketide producers have high G+Ccodons; thus, yeast may have low amounts of some tRNAs needed for PKSgene expression. The correction of these deficiencies is described inthis example, and the invention also provides modified yeast host cellsuseful to facilitate analysis of success.

Other case-by-case potential issues with yeast include the possibilitythat some polyketide products may be toxic or may require additionalmodifications for maturation (e.g. glycosylation, P450 hydroxylation).Several methods provided by the invention may be taken to circumventthese issues should they arise. For toxicity, production may becontrolled to occur in stationary phase growth (as with 6-MSAproduction); resistance factors from the wild type host may beintroduced into the yeast host (e.g. methylation of ribosomes for someantibiotics); a non-toxic-precursor to the polyketide may be producedand converted ex vivo (e.g. produce 6-dEB in one strain and convert itto erythromycin in another), and others. Additional modifications to thepolyketide may be accomplished by cloning and expressing modificationenzymes into the host strain, chemical or enzymic transformation, and/orbiosynthetic transformation in a second strain (e.g. convert 6-dEBanalogs to erythromycin analogs by feeding 6-dEB to a Streptomyces orSaccharopolyspora strain capable of glycosylation and P450hydroxylation).

Most modular PKSs require either or both malonyl-CoA or(2S)-methylmalonyl-CoA as a source of 2-carbon units for polyketidebiosynthesis. The malonyl-CoA pools in yeast are quite sufficient forpolyketide synthesis, as illustrated by the production of large amountsof 6-MSA in yeast. However, S. cerevisiae does not produce(2S)-methylmalonyl-CoA and does not possess biosynthetic pathways formethylmalonyl-CoA biosynthesis. Hence, a heterologous biosyntheticpathway must be introduced into S. cerevisiae to support biosynthesis ofpolyketides that use (2S)-methylmalonyl-CoA as a precursor.

There are three routes or biosynthetic pathways for the synthesis ofmethylmalonyl-CoA that can be engineered into yeast, as shown in FIG. 5.These pathways have been shown to produce methylmalonyl-CoA in E. coliand can be used to produce methylmalonyl-CoA in yeast. This exampledescribes the identification of a system for methylmalonyl-CoAproduction in yeast, and a method for introducing it into the yeastchromosome.

The vitamin B12-dependent methylmalonyl-CoA mutase pathway produces(2R)-methylmalonyl-CoA from succinyl-CoA. The (2R)-methylmalonyl-CoA isconverted to the (2S)-diastereomer via methylmalonyl-CoA epimerase, asshown above. These enzymes are present in a variety of organisms, butnot yeast; BLAST searches of the available genomic databases reveals atleast 10 methylmalonyl-CoA mutases and 10 methylmalonyl-CoA epimerasesin various organisms. The Propionibacterium shermanii methylmalonyl-CoAmutase has been expressed in E. coli as the apo-enzyme, which requiresaddition of vitamin B12 for in vitro activity (McKie, N., et al.,Adenosylcobalamin-dependent methylmalonyl-CoA mutasefromPropionibacterium shermanii. Active holoenzyme producedfrom Escherichiacoli. Biochem J, 1990. 269(2): p. 293-8; incorporated herein byreference). By use of a medium that enables uptake of the vitamin B12precursor hydroxocobalamin (Amaratunga, M., et al., A synthetic modulefor the metH gene permits facile mutagenesis of the cobalamin-bindingregion of Escherichia coli methionine synthase: initial characterizationof seven mutant proteins. Biochemistry, 1996. 35(7): p. 2453-63;incorporated herein by reference), and in accordance with the methods ofthe invention, one can express active P. shermanii methylmalonyl-CoAmutase holoenzyme in E. coli and produce (2R)-methylmalonyl-CoA in suchcells. In addition, one can employ the single subunit methylmalonyl-CoAmutase from E. coli. The present invention also provides the genesencoding methylmalonyl-CoA epimerase from B. subtilis, P. shermanii andS. coelicolor and methods for using them in converting(2R)-methylmalonyl-CoA to the needed (2S)-diastereomer. A preferredmethod is to express in yeast the methylmalonyl-CoA mutase from E. coli,because it is a single ORF, and necessary codons are plentiful in yeast.Alternatively, the P. shermanii enzyme can be used.

PCC catalyzes the biotin-dependent carboxylation of propionyl-CoA toproduce (2S)-methylmalonyl-CoA, as shown above; the pathway alsoincludes a biotin carrier proteinibiotin carboxylase. In S. coelicolor,Rodriguez and Gramajo identified genes for PCC (pccB) and a biotincarrier proteinibiotin carboxylase (accA1) (Rodriguez, E. and H.Gramajo, Genetic and biochemical characterization of the alpha and betacomponents ofapropionyl-CoA carboxylase complex of Streptomycescoelicolor A3(2). Microbiology, 1999. 145(Pt 11)): p. 3109-19;incorporated herein by reference). Introduction into E. coli of S.coelicolor pccB and accA1 along with propionyl-CoA ligase (as a supplyof propionyl-CoA), results in the production of methylmalonyl-CoA inthat organism. A search of the genomic database reveals B. subtilis asan additional source of the enzymes involved in the PCC pathway.

In one embodiment of the invention, one can express the S. coelicolorpccB and accA1 in yeast, because these are expressed and the proteinsare functional in E. coli. Should codon usage prove suboptimal whenexpressing the S. coelicolor genes in yeast, homologs from B. subtiliscan be employed. Should the levels of propionyl-CoA be suboptimal forPCC, one can co-express a propionyl-CoA ligase in the yeast host.Intracellular propionyl-CoA can be greatly increased in E. coli byexpressing the Salmonella propionyl-CoA ligase, PrpE, and supplementingthe growth media with propionate, as described below.

An additional method for the production of (2S)-methylmalonyl-CoAprovided by the present invention utilizes the matB and matC genes fromRhizobium (An, J. H. and Y. S. Kim, A gene cluster encoding malonyl-CoAdecarboxylase (MatA), malonyl-CoA synthetase (MatB) and a putativedicarboxylate carrier protein (MatC) in Rhizobium trifolii—cloning,sequencing, and expression of the enzymes in Escherichia coli. Eur JBiochem, 1998. 257(2): p. 395-402; incorporated herein by reference) orS. coelicolor (see schematic above). The matABC genes code for abiosynthetic pathway that converts malonate to acetyl-CoA throughformation of malonyl-CoA via MatB and subsequent decarboxylation byMatA. MatB, the malonyl-CoA ligase, also accepts methylmalonate as asubstrate (An, J. H. and Y. S. Kim, A gene cluster encoding malonyl-CoAdecarboxylase (MatA), malonyl-CoA synthetase (MatB) and a putativedicarboxylate carrier protein (MatC) in Rhizobium trifolii—cloning,sequencing, and expression of the enzymes in Escherichia coli. Eur JBiochem, 1998. 257(2): p. 395-402; incorporated herein by reference) andcatalyzes formation of methylmalonyl-CoA. The substrates malonate ormethylmalonate enter the cell through a diacid transporter, the productof the matC gene. Khosla et al. have shown that when E. coli containingthe Rhizobium matBC is fed (2R,2S)-mEthylmalonate,(2R,2S)-methylmalonyl-CoA is produced. Furthermore, when an S.coelicolor strain expressing the genes for the synthesis of thepolyketide aglycone, 6-deoxyeythronolide B (6-dEB), and containingRhizobium matBC, is fed methylmalonate, a 3-fold increase in productionof 6-dEB is observed. In accordance with the methods of the invention,one can express the matB and matC genes from Rhizobium in yeast, becausethese are expressed and the proteins are functional in E. coli and S.coelicolor, or, alternatively the matBC genes from S. coelicolor.

Active PKSs require post-translational phosphopantetheinylation at eachACP of each module, but yeast does not contain a P-pant transferase withthe needed specificity (Kealey, J. T., et al., Production of apolyketide natural product in nonpolyketide-producing prokaryotic andeukaryotic hosts. Proc Natl Acad Sci USA, 1998. 95(2): p. 505-9;incorporated herein by reference). Previous work (Kealey, J. T., et al.,Production of a polyketide natural product in nonpolyketide-producingprokaryotic and eukaryotic hosts. Proc Natl Acad Sci USA, 1998. 95(2):p. 505-9; incorporated herein by reference) has shown that introductionof the B. subtilis P-pant transferase gene, sfp, into yeast results inan expressed Sfp capable of modifying an iterative PKS, 6-MSAS. Gokhaleet al. demonstrated that the ACP domains in the DEBS PKS are substratesfor Sfp, so Sfp is a general modifying enzyme for PKSs (Gokhale, R. S.,et al., Dissecting and exploiting intermodular communication inpolyketide synthases. Science, 1999. 284(5413): p. 482-5; incorporatedherein by reference). In preferred yeast host cells of the invention,the sfp gene is inserted into a neutral site of the yeast chromosome.

In developing a system to produce polyketides and optimize fermentationprocedures, the ability to measure intracellular concentrations ofsubstrates (i.e. acyl-CoAs) and of the PKS is beneficial. In most cells,CoA esters are not present in sufficient amounts to allow directmeasurement by HPLC using ultraviolet detection or other simple methodsof detection. In E. coli, the method of choice to quantify CoA pools isto feed [³H] β-alanine to a mutant deficient in aspartate decarboxylase(PanD), which cannot produce endogenous β-alanine (Jackowski, S, and C.O. Rock, Regulation of coenzyme A biosynthesis. J Bacteriol, 1981.148(3): p. 926-32; incorporated herein by reference). The PanD strainincorporates about ten-fold more radioactivity into CoA pools than doeswild type E. coli. Because β-alanine is a direct precursor of CoA, theradioactive label enters the CoA pool without dilution, and acyl-CoAscan be separated on HPLC and quantified by radioactivity measurement.Because there is no radioisotope dilution, the radioactivity measuredreflects exact intracellular concentrations of the acyl-CoAs.

BLAST searches did not reveal an E. coli PanD homolog in the yeastgenome; however, yeast may be a β-alanine or pantothenate auxotroph.Indeed, for CoA biosynthesis, yeast requires either exogenouspantothenate, which enters the cell via the Fen2p transporter, orexogenous β-alanine, which enters via the general amino acid permease(Gap1p) (Stolz, J. and N. Sauer, The fenpropimorph resistance gene FEN2from Saccharomyces cerevisiae encodes a plasma membrane H+-pantothenatesymporter. J Biol Chem, 1999. 274(26): p. 18747-52; incorporated hereinby reference). [³H] β-alanine is incorporated into CoA pools of yeast(see below), but it is presently unknown whether isotope dilution occursdue to endogenous β-alanine production by some unknown pathway. Thus, toenable quantitation, one can determine the specific activity of CoApools in yeast labeled with exogenous [³H] β-alanine. Cells producingpolyketides generally express low levels of high molecular weight PKSsthat are barely detectable on SDS-PAGE using protein stains. The abilityto label CoA with [³H] β-alanine can also be used to quantify a PKSexpressed in the host cells because the phosphopantetheine moiety of CoAcontaining β-alanine is transferred to the ACP domain in each module ofa PKS. Thus, knowing the specific activity of labeled intracellularCoAs, a PKS can be simply quantified by radioactivity after SDS-PAGE.

The G+C content of most PKS genes is in the range of 60 to 70%, whilethat of yeast genes is 40%. Thus, some tRNAs needed to translate PKSgenes are scarce (but not absent) in yeast. However, many genes withhigh G+C content have been expressed in yeast. As examples, the large(1560 bp) DHFR-TS gene from Leishmania major (63% G+C) is expressed wellin yeast, despite the fact that it contains several codons rarely usedin yeast (Grumont, R., W. Sirawaraporn, and D. V. Santi, Heterologousexpression of the bifunctional thymidylate synthase-dihydrofolatereductase from Leishmania major. Biochemistry, 1988. 27(10): p. 3776-84;incorporated herein by reference). Moreover, as mentioned below, the PKS6-MSAS (G+C=58%) is also expressed well in yeast (Kealey, J. T., et al.,Production of a polyketide natural product in nonpolyketide-producingprokaryotic and eukaryotic hosts. Proc Natl Acad Sci USA, 1998. 95(2):p. 505-9; incorporated herein by reference). Thus, one can demonstratethe general applicability of a yeast expression system without initialconcern for potential codon usage problems. Nevertheless, if a desiredPKS does not express well in yeast, the present invention providesseveral methods to solve a “codon usage” problem observed with aparticular polyketide.

First, one can change the codons at the 5′ end of the gene to reflectthose more frequently found in yeast genes. Batard et al. (Batard, Y.,et al., Increasing expression of P450 and P450-reductase proteins frommonocots in heterologous systems [In Process Citation]. Arch BiochemBiophys, 2000. 379(1): p. 161-9; incorporated herein by reference)successfully employed a similar method to express in yeast wheat genesfor a P450 and P450 reductase with high G+C content (56%) and strongbias of codon usage unfavorable to yeast. Another method is to introduceyeast tRNA genes with anti-codons modified to represent codons common inPKS sequences. A similar method has been successfully used in E. coli toenhance expression of high G+C genes (Carstens, C.-P., et al., NewBL21-CodonPlus™ Cells Correct Codon Bias in GC-Rich Genomes. StrategiesNewsletter from Stratagene Corp., 2000. 13(1): p. 31-33; incorporatedherein by reference), including PKS genes from Actinomycetes. A thirdmethod is to synthesize chemically the gene with codons optimized forexpression in yeast. The cost for contract synthesis of a 30,000 bp gene(e.g. ˜6-module PKS), including sequence verification, is approximately$3 per base, or about $100,000. For a valuable product (e.g.epothilone), the cost is not prohibitive.

In an illustrative embodiment of the invention, a yeast strain deficientin Ura, Trp, His and Leu biosynthesis is employed as a host to allowselection of plasmids containing these markers. This host is modified inaccordance with the methods of the invention by introducing genes thatproduce the needed methylmalonyl-CoA substrate and P-pant transferasefor post-translational modifications of PKSs. These are preferablyintegrated into the yeast chromosome, because they are necessary forproduction of any polyketide. To validate functional expression of thesubstrate genes, one can measure methylmalonyl-CoA pools. For validationof P-pant transferase activity, one can coexpress 6-MSAS and measure[³H] phosphopantetheinylation of the enzyme as well as 6-MSA production.Should either be deficient, one can increase gene copy number.

For PKS gene expression, one can use replicating vectors based on the 2micron replicon, because plasmids may have to be rescued for analysisshould a problem arise. A typical modular PKS gene cluster (eg. 3 ORFS,˜10 kB each, as in erythromycin) can be introduced on three or morevectors; such plasmids (containing Ura, Trp and Leu markers) areavailable and similar to those used in the studies of 6-MSAS expressionin yeast. A PKS consisting of three large proteins can be functionallyreconstituted from separately expressed genes (Xue, Q., et al., Amultiplasmid approach to preparing large libraries of polyketides. ProcNatl Acad Sci USA, 1999. 96(21): p. 11740-5; incorporated herein byreference). Once a system is established for a particular PKS ofinterest, one can integrate the PKS genes into stable, neutral sites ofthe chromosome.

Preferred promoters include the glucose repressible alcoholdehydrogenase 2 (ADH2) promoter and the galactose-inducible (GAL1)promoter. The former has been used to produce high amounts of thepolyketide 6-MSA in yeast, and the latter is highly controllable bygalactose in the medium.

A model modular PKS that one can use to optimize the yeast host is thewell studied DEBS1. In this model system, the first ORF of the modularPKS for erythromycin biosynthesis (DEBS1) has been fused to athioesterase domain (TE) and produces a readily detectable triketidelactone when expressed in S. coelicolor, and more recently E. coli (Kao,C. M., et al., Engineered biosynthesis of a triketide lactone from anincomplete modular polyketide synthase. J. Am. Chem. Soc., 1994.116(25): p. 11612-11613; Cortes, J., et al., Repositioning of a domainin a modular polyketide synthase to promote specific chain cleavage.Science, 1995. 268(5216): p. 1487-9; both incorporated herein byreference). The gene contains 2 PKS modules, is about 12 kB, andproduces a protein that is 300 kDa. This model allows one to optimizethe engineered host for acyl-CoA levels and post-translationalmodifications, the PKS for G+C content, and to develop the neededanalytical methods. Once optimized for DEBS1, one can express any givenmodular PKS.

Previously, it has been shown that the fungal gene encoding6-methylsalicylic acid synthase (6-MSAS) from Penicillium patulum wasexpressed in S. cerevisiae and E. coli and the polyketide6-methylsalicylic acid (6-MSA) was produced (Kealey, J. T., et al.,Production of a polyketide natural product in nonpolyketide-producingprokaryotic and eukaryotic hosts. Proc Natl Acad Sci USA, 1998. 95(2):p. 505-9; incorporated herein by reference). In both bacterial and yeasthosts, polyketide production required co-expression of 6-MSAS and aheterologous phosphopantetheinyl transferase (Sfp), which was requiredto convert the expressed apo-PKS to the holo-enzyme. Production of 6-MSAin E. coli was both temperature- and glycerol-dependent and levels ofproduction (˜60 mg/L) were lower than those of the native host, P.patulum. In yeast, the 6-MSAS and sfp genes were co-expressed fromseparate replicating plasmids, and gene expression was driven by theglucose repressible alcohol dehydrogenase 2 (ADH2) promoter. In anon-optimized shake flask fermentation, the yeast system produced 6-MSAat levels of 2,000 mg/L. This was the first report of expression of anintact PKS gene in yeast or E. coli, and demonstrated thatextraordinarily high levels of polyketides can be produced in yeast.

Previously, a two vector system was developed for heterologousexpression of the three genes comprising the DEBS polyketide genecluster (Ziermann, R., Betlach, M., A Two-vector System for theProduction of Recombinant Polyketides in Streptomyces. J. Ind.Microbiol. Biotech., 2000, 24:46-50; incorporated herein by reference).Individual DEBS genes and pairwise combinations of two such genes wereeach cloned downstream of the actinorhodin (acti) promoter in twocompatible Streptomyces vectors: the autonomously replicating vector,pKAO127′Kan′, and the integrating vector, pSET152. When the resultingplasmids were either simultaneously or sequentially transformed into theheterologous host, Streptomyces lividans K4-114, the polyketide product,6-dEB, was produced. This work showed that the DEBS genes could be splitapart and expressed on separate plasmids, and that efficienttrans-complementation of modular polyketide synthase subunit proteinsoccurred in the heterologous host.

A three-plasmid system for heterologous expression of DEBS has beendeveloped to facilitate combinatorial biosynthesis of polyketides madeby type I modular PKSs (Xue, Q., et al., A multiplasmid approach topreparing large libraries of polyketides. Proc Natl Acad Sci USA, 1999.96(21): p. 11740-5; incorporated herein by reference). The eryA PKSgenes encoding the three DEBS subunits were individually cloned intothree compatible Streptomyces vectors carrying mutually selectableantibiotic resistance markers. A strain of Streptomyces lividanstransformed with all three plasmids produced 6-dEB at a level similar tothat of a strain transformed with a single plasmid containing all threegenes. The utility of this system in combinatorial biosynthesis wasdemonstrated through production of a large library of greater than 60modified polyketide macrolactones, using versions of each plasmidconstructed to contain defined mutations. Combinations of these vectorsets were introduced into S. lividans, resulting in strains producing awide range of 6-dEB analogs. This method can be extended to any modularPKS and has the potential to produce thousands of novel naturalproducts, including ones derived from further modification of the PKSproducts by tailoring enzymes. Moreover, the ability to express themodular PKSs (such as DEBS) from three separate plasmids providesadvantages in the commercialization of polyketide production byheterologous expression of modular PKSs in yeast and E. coli inaccordance with the methods of the present invention.

As described in Example 1, the translationally coupled genes, mutA andmutB, encoding the β- and α-subunits of methylmalonyl-CoA mutase fromPropionibacterium shermanii, were amplified by PCR and inserted into anE. coli expression vector containing a T-7 promoter. The naturallyoccurring GTG start codon for mutB was changed to ATG to facilitateexpression (Amaratunga, M., et al., A synthetic module for the metH genepermits facile mutagenesis of the cobalamin-binding region ofEscherichia coli methionine synthase: initial characterization of sevenmutant proteins. Biochemistry, 1996. 35(7): p. 2453-63; incorporatedherein by reference). Heterologous expression of the mutase genes inmedia containing [³H] β-alanine and the adenosylcobalamin (coenzyme B₁₂)precursor, hydroxocobalamin, yielded active methylmalonyl-CoA mutase.HPLC analysis of extracts from E. coli BL21(DE3)/panD harboring themutase genes indicated production of methylmalonyl-CoA, which comprised13% of the intracellular CoA pool (shown in FIG. 6). This workdemonstrates that one can introduce a biosynthetic pathway for animportant PKS substrate into a heterologous host, and that one canmeasure the intracellular concentration of acyl-CoAs. In accordance withthe present inveniton, the methylmalonyl-CoA mutase gene (sbm) from E.coli, which has codon usage closer to yeast and encodes a singlepolypeptide (Haller, T., et al., Discovering new enzymes and metabolicpathways: conversion of succinate to propionate by Escherichia coli.Biochemistry, 2000. 39(16): p. 4622-9; incorporated herein byreference), can also be employed.

FIG. 6 shows acyl-CoA analysis of E. coli overexpressingmethylmalonyl-CoA mutase. The level of ³H detected in fractionscollected from HPLC of cell-free extracts from [³H] β-alanine-fed E.coli harboring either the pET control vector (solid trace) or pEToverexpressing the mutase (dashed trace) is shown.

As described in Example 1, methylmalonyl-CoA epimerase was purified fromPropionibacterium shermanii and N-terminal and internal protein sequencewas obtained. Degenerate PCR primers based on the amino acid sequenceswere designed and were used to amplify a 180 bp PCR product from P.shermanii genomic DNA. The PCR product was labeled and used to isolatethe epimerase gene from P. shermanii. The methylmalonyl-CoA epimerasegenes from B. subtilis (Haller, T., et al., Discovering new enzymes andmetabolic pathways: conversion of succinate to propionate by Escherichiacoli. Biochemistry, 2000. 39(16): p. 4622-9; incorporated herein byreference), and S. coelicolor can also be employed in the methods of thepresent invention.

Propionyl-CoA is not detected in E. coli SJ16 cells grown in thepresence of [³H] β-alanine with or without the addition of propionate inthe growth media. When E. coli SJ16 cells were transformed with apACYC-derived plasmid containing the Salmonella typhimuriumpropionyl-CoA ligase gene (prpE) under the control of the lac promoter,a small amount of propionyl-CoA was observed (˜0.2% of total CoA pool)in cell extracts. When 5 mM sodium propionate was included in theculture medium, about 14-fold more propionyl-CoA was produced (˜3% ofthe total CoA pool).

FIG. 7 shows acyl-CoA analysis in S. cerevisiae. The level of ³Hdetected in fractions collected from HPLC of cell-free extracts from[³H] β-alanine-fed S. cerevisiae after growth for 24 hours (solidtrace), 48 hours (dashed trace) and 66 hours (dotted trace) is shown.The yeast strain InvScl (Kealey, J. T., et al., Production of apolyketide natural product in nonpolyketide-producing prokaryotic andeukaryotic hosts. Proc Natl Acad Sci USA, 1998. 95(2): p. 505-9;incorporated herein by reference), grown in synthetic YNB media lackingpantothenate and β-alanine, was used for acyl-CoA analysis. Yeastcultures starved of β-alanine were fed [³H] β-alanine and the cultureswere grown for 24, 48 and 66 hours at 30° C. Cells were disrupted withglass beads in the presence of 10% cold TCA and acyl-CoAs were separatedby HPLC and quantified by scintillation counting. The yeast CoA poolswere labeled with [³H], but the extent of isotope dilution remainsunclear. One can measure the specific activity of total CoA in thesestrains to ascertain the extent of isotope dilution.

For PKS genes and initial studies of metabolic pathway genes, one canemploy the analogous sets of bluescript cloning vectors and yeast 2micron replicating shuttle vectors used in 6-MSA production (Kealey, J.T., et al., Production of a polyketide natural product innonpolyketide-producing prokaryotic and eukaryotic hosts. Proc Natl AcadSci USA, 1998. 95(2): p. 505-9; incorporated herein by reference). Withthese vectors, yeast expression is driven by the alcohol dehydrogenase 2(ADH2) promoter, which is tightly repressed by glucose and is highlyactive following glucose depletion that occurs after the culture reacheshigh density. Both vector sets have a “common cloning cassette” thatcontains, from 5′ to 3′, a polylinker (L1), the ADH2 (or other)promoter, a Nde I restriction site, a polylinker (L2), an ADH2 (orother) terminator, and a polylinker (L3). Due to excess restrictionsites in the yeast shuttle vectors, genes of interest are firstintroduced into intermediate bluescript cloning vectors via the Nde Isite, to generate the ATG start codon, and a downstream restriction sitein the L2 polylinker that is common to the bluescript and yeast shuttlevectors (shown in FIG. 8). The promoter-gene cassette is then excised asan L1-L2 fragment and transferred to the yeast expression vectorcontaining the transcriptional terminator.

Host strains for model systems include commonly available yeast strainswith nutritional deficiencies (Ura, Trp, His, Leu) that can harbor atleast three replicating vectors (see below). If it is necessary toexpress more than three PKS genes simultaneously, one can clone multiplepromoter-PKS gene-terminator cassettes into the same vector or use afourth replicating vector with a different nutritional marker (i.e. Leu)or an antibiotic marker (i.e. G418). One can also construct an analogousset of bluescript cloning and yeast expression/shuttle vectorscontaining a galactose-inducible promoter. The galactose promoter-Gal4activator system is more tightly regulated than the ADH2 promoter, andmay be beneficial or necessary for expression of proteins that are toxicto yeast (Mylin, L. M., et al., Regulated GAL4 expression cassetteproviding controllable and high-level outputfrom high-copy galactosepromoters in yeast. Methods Enzymol, 1990. 185: p. 297-308; incorporatedherein by reference).

Genes involved in the production of substrates (eg. methylmalonyl-CoAand/or propionyl-CoA), and the sfp gene can preferably be stablyintegrated into the yeast chromosome in appropriate copy number toproduce adequate levels of desired acyl-CoAs and post translational PKSmodifications. Genes can first be introduced into the intermediatebluescript cloning vector as described. Then, the fragment containingthe promoter-gene-terminator cassette can be transferred as a L1-L3fragment to a yeast “delta integration” vector (Lee, F. W. and N. A. DaSilva, Improved efficiency and stability of multiple cloned geneinsertions at the delta sequences of Saccharomyces cerevisiae. ApplMicrobiol Biotechnol, 1997. 48(3): p. 339-45; Lee, F. W. and N. A. DaSilva, Sequential delta-integration for the regulated insertion ofcloned genes in Saccharomyces cerevisiae. Biotechnol Prog, 1997. 13(4):p. 368-73; both incorporated herein by reference) that allowschromosomal integration of the cassettes into one or more of the ca. 425delta sequences dispersed throughout the yeast chromosome (see FIG. 9).These vectors have cloning sites compatible with those in the L1-L3linkers to permit direct transfer of promoter-gene-terminator cassettesas L1-L3 fragments. They also contain the excisable Ura3 selectionmarker flanked by two bacterial hisG repeats (“URA Blaster”), enablinginsertion of multiple identical or different genes into the yeastchromosome by repetitive integrations. After selection for geneintegration on media lacking uracil, the Ura3 gene fragment is removedby selecting for marker loss via excisional recombination by positiveselection with 5-fluoroorotic acid (FOA), which renders the Ura3 genetoxic to yeast. This enables the introduction of stable pathways neededfor acyl-CoA precursors and Sfp into yeast, while conserving the Uramarker to allow its subsequent use in plasmids containing other genes.

The single-gene mutase, Sbm (Sleeping beauty mutase), from E. coli(Haller, T., et al., Discovering new enzymes and metabolic pathways:conversion of succinate to propionate by Escherichia coli. Biochemistry,2000. 39(16): p. 4622-9; incorporated herein by reference), can becloned as follows. Primers designed based on the DNA sequence were usedto PCR amplify the sbm gene from E. coli genomic DNA as a NdeI-L2fragment. The general strategy for cloning the genes into yeastexpression vectors follows that of Kealey et al. (Kealey, J. T., et al.,Production of a polyketide natural product in nonpolyketide-producingprokaryotic and eukaryotic hosts. Proc Natl Acad Sci USA, 1998. 95(2):p. 505-9; incorporated herein by reference) (see FIG. 9). One can firstclone the genes as NdeI-L2 fragments into the intermediate bluescriptcloning vector. The promoter-gene-terminator cassette can then beexcised as an L1-L3 fragment, transferred to the yeast integratingvector, restricted with L1/L3, and introduced into the yeast chromosomeas described above. As an alternative to Sbm, one can use the two-genemutase from P. shermanii; the translationally coupled genes have eachbeen amplified by PCR as NdeI-L2 fragments and can be integrated intoyeast as described above.

The genes encoding matABC have been cloned into a bluescript vector (An,J. H. and Y. S. Kim, A gene cluster encoding malonyl-CoA decarboxylase(MatA), malonyl-CoA synthetase (MatB) and a putative dicarboxylatecarrier protein (MatC) in Rhizobium trifolii—cloning, sequencing, andexpression of the enzymes in Escherichia coli. Eur J Biochem, 1998.257(2): p. 395-402; incorporated herein by reference). One can isolatethe matB (methylmalonyl-CoA ligase) and matC (dicarboxylic acidtransporter) genes by PCR, each as a NdeI-L2 fragment, and integratethem into the yeast chromosome as described above and shown in theschematic below. Yeast transformed with matBC will be treated withmethylmalonic acid, and cells extracts can be analyzed formethylmalonyl-CoA.

The pccB and accA1 genes involved in the propionyl-CoA carboxylationpathway in S. coelicolor can be amplified by PCR from genomic DNA. Asshown in FIG. 9, the genes can be cloned into the intermediatebluescript vector between Nde I and L2, then transferred to the yeastintegrating vector via L1/L3. One can express the S. coelicolor genesshown to be effective in E. coli; should codon usage be suboptimal, onecan employ the B. subtilis orthologs (discussed above).

FIG. 9 shows a general method for cloning genes into yeast expressionvectors.

In one embodiment, the recombinant yeast host cells of the inventionco-express the B. subtilis P-pant transferase, Sfp, with a PKS toconvert the apo PKS to its holo form. The sfp gene is available onBluescript™ (Stratagene) cloning and yeast shuttle/expression vectorsand is functional in yeast (Kealey, J. T., et al., Production of apolyketide natural product in nonpolyketide-producing prokaryotic andeukaryotic hosts. Proc Natl Acad Sci USA, 1998. 95(2): p. 505-9;incorporated herein by reference), so one can simply construct stablestrains expressing this gene. One to several copies (as determinedoptimal) of the sfp gene can be introduced into delta sequences in theyeast chromosome as described above. One can test the activity of theintegrated sfp gene by co-expressing 6-MSAS on a replicating vector, bymeasuring the Sfp-dependent 6-MSA production (Kealey, J. T., et al.,Production of a polyketide natural product in nonpolyketide-producingprokaryotic and eukaryotic hosts. Proc Natl Acad Sci USA, 1998. 95(2):p. 505-9; incorporated herein by reference), and by quantifying theincorporation of [³H] β-alanine into the ACP domain of the PKS (seebelow). This allows one to determine the optimal number of copies of thesfp gene needed for maximal polyketide production.

The gene for the modular PKS, DEBS1+TE, is available as a NdeI-EcoRIfragment, which can be readily introduced into a yeastshuttle/expression vector as indicated in FIG. 9. Yeast strainsexpressing DEBS1+TE are analyzed for the [³H]-phosphopantetheinylationof the PKS, and for production of triketide lactone by liquidchromatography/mass spectrometry.

³H labeling of intracellular Acyl-CoAs is carried out as follows. Cellsare treated with [³H] β-alanine (available at 50 Ci/mmol) in definedmedia lacking pantothenate, enabling the radioactive precursor ofpantothenate to enter the CoA pool. Cells are then disrupted, CoA estersare separated by HPLC, and the radioactivity quantified by liquidscintillation counting, as described above.

Saccharomyces cerevisiae host cells are grown, and extracts prepared asfollows. Defined minimal YNB media (1 mL) lacking pantothenate butcontaining 1 μM β-alanine are inoculated with a single colony of S.cerevisiae (InvScl, or Fen2b deletion strain) from a YPD plate. Theculture is grown to stationary phase and 10 μl of the stationary cultureare used to inoculate the above media lacking β-alanine andpantothenate. The culture is incubated for 4 hours and 10 μl of the“starved” culture is used to inoculate media (1 mL) containing 10 μCi[³H] β-alanine (50 Ci/mmol; 0.2 mM final β-alanine). After culturegrowth for appropriate times, the cells from a 1 mL culture arecollected by centrifugation and washed with water. The cells aresuspended in 200 μl of 10% cold trichloroacetic acid (TCA), containingstandard unlabeled acyl-CoAs as chromatography markers (malonyl-,methylmalonyl-, succinyl-, acetyl-, propionyl-CoA, and CoA). The cellsare disrupted by vortexing with glass beads, and the supernatentanalyzed by HPLC.

HPLC is performed using a 150×4.6 mm 5μ ODS-3 INERTSIL HPLC columnpurchased from Metachem technology. HPLC buffer A is 100 mM sodiumphosphate monobasic, 75 mM sodium acetate, pH 4.6 and buffer B is 70%buffer A, 30% methanol. The HPLC column is equilibrated at 10% buffer Bat a flow rate of 1 mL/min. Following injection, a linear gradient to40% buffer B is implemented over 35 minutes, followed by a lineargradient to 90% buffer B over 20 minutes. The gradient affords base-lineseparation of the standard acyl-CoAs. The eluant is monitored at 260 nmand fractions are collected and counted in a scintillation counter.

Determination of the specific activity of the total CoA pool is carriedout as follows. S. cerevisiae cultures are labeled with 100 μCi of [³H]β-alanine as described above. The yeast cells are disrupted and theextract is treated with 100 μM hydroxylamine, pH 8.5, to convert allacyl-CoAs to CoA. The labeled CoA is isolated by HPLC as described aboveand converted to acetyl-CoA with E. coli acetyl-CoA synthase (Sigma),using [¹⁴C]-acetate as a substrate. The [³H, ¹⁴C]-acetyl-CoA isseparated by HPLC and the dual labels quantified by scintillationcounting. The mmol CoA is determined by ¹⁴C, and specific activity ofCoA determined from the ³H dpm per mmol CoA. The isotope dilution,reflecting endogenous production of β-alanine, is calculated by thespecific activity of [³H] CoA/specific activity [³H] β-alanine used inthe test.

Analysis of PKS expression levels is carried out as follows. Each ACPdomain of each module of an active PKS is post-translationally modifiedwith phosphopantetheine derived from CoA. Using yeast cells treated with[³H] β-alanine (described above), one can label the PKS with highspecific activity tritium. The protein will be separated on SDS-PAGE,eluted and radioactivity determined by liquid scintillation counting.

EXAMPLE 3 Conversion of Erythronolides to Ervthromycins

A sample of a polyketide (˜50 to 100 mg) is dissolved in 0.6 mL ofethanol and diluted to 3 mL with sterile water. This solution is used tooverlay a three day old culture of Saccharopolyspora erythraea WHM34 (aneryA mutant) grown on a 100 mm R2YE agar plate at 30° C. After drying,the plate is incubated at 30° C. for four days. The agar is chopped andthen extracted three times with 100 mL portions of 1% triethylamine inethyl acetate. The extracts are combined and evaporated. The crudeproduct is purified by preparative HPLC (C-18 reversed phase,water-acetonitrile gradient containing 1% acetic acid). Fractions areanalyzed by mass spectrometry, and those containing pure compound arepooled, neutralized with triethylamine, and evaporated to a syrup. Thesyrup is dissolved in water and extracted three times with equal volumesof ethyl acetate. The organic extracts are combined, washed once withsaturated aqueous NaHCO₃, dried over Na₂SO₄, filtered, and evaporated toyield ˜0.15 mg of product. The product is a glycosylated andhydroxylated compound corresponding to erythromycin A, B, C, and D butdiffering therefrom as the compound provided differed from 6-dEB.

EXAMPLE 4 Measurement of Antibacterial Activity

Antibacterial activity is determined using either disk diffusion assayswith Bacillus cereus as the test organism or by measurement of minimuminhibitory concentrations (MIC) in liquid culture against sensitive andresistant strains of Staphylococcus pneumoniae.

EXAMPLE 5 Evaluation of Antiparasitic Activity

Compounds can initially screened in vitro using cultures of P.falciparum FCR-3 and

K1 strains, then in vivo using mice infected with P. berghei. Mammaliancell toxicity can be determined in FM3A or KB cells. Compounds can alsobe screened for activity against P. berhei. Compounds are also tested inanimal studies and clinical trials to test the antiparasitic activitybroadly (antimalarial, trypanosomiasis and Leishmaniasis).

The invention having now been described by way of written descriptionand example, those of skill in the art will recognize that the inventioncan be practiced in a variety of embodiments and that the foregoingdescription and examples are for purposes of illustration and notlimitation of the following claims.

1. A method for converting (R)-methylmalonyl CoA to (S)-methylmalonylCoA in a host cell, the method comprising culturing the host cell ofclaim 22 under conditions such that said epimerase is expressed, and(R)-methylmalonyl CoA is converted into (S)-methylmalonyl CoA.